Use of pertussis toxin as a therapeutic agent

ABSTRACT

The present application relates to the use of pertussis toxin, and its derivatives, analogs, salts and pharmaceutical equivalents. In one embodiment, the invention provides a method of treating or preventing a neurological disease or injury by administering pertussis toxin to the individual.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/782,151, filed on Oct. 12, 2017, which is a continuation-in-part ofand claims priority to U.S. Pat. No. 9,801,921, filed on Dec. 12, 2013and issued on Oct. 31, 2017, which is the National Phase ofInternational Application PCT/US2012/045065, filed Jun. 29, 2012, whichclaims priority to U.S. Provisional Application Ser. No. 61/503,491,which was filed on Jun. 30, 2011, the entire contents of each of thesereferences are incorporated herein by reference for all purposes.

FIELD OF THE INVENTION

The invention relates generally to the field of medicine and neurologyand autoimmunity and, more specifically, to pertussis toxin and methodsof treating and preventing neurological and autoimmune diseases andconditions such as multiple sclerosis and stroke (e.g., ischemicstroke).

BACKGROUND

All publications herein are incorporated by reference to the same extentas if each individual publication or patent application was specificallyand individually indicated to be incorporated by reference. Thefollowing description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed invention, or that any publication specifically orimplicitly referenced is prior art.

Multiple sclerosis (MS), or disseminated sclerosis or encephalomyelitisdisseminata is a neurological and autoimmune disease where myelinsheaths around axons of the brain and spinal cord are damaged. Theresult is difficulty for nerve cells in the brain and spinal cord toeffectively communicate with each other. Various neurological symptomscan occur, often progressing into physical and cognitive disability,until often permanent neurological problems occur as the diseaseadvances. The disease affects 2.5 million people and life expectancy ofthose with MS is about 5 to 10 years lower than the normal population.At present, the exact cause of MS is unknown, although experimentalautoimmune encephalomyelitis (EAE) is the primary animal model used tostudy MS. Unfortunately, there is no known cure for MS, and MSmedications often have adverse effects. Thus, there is a great need inthe art for novel and effective treatments for neurological andautoimmune diseases such as multiple sclerosis.

Similarly, stroke is a leading cause of disability and death in theUnited States and other developed countries. Advances in theunderstanding of the pathophysiology of stroke have generated a plethoraof new investigative studies in the treatment of stroke; however therehas been little success in the clinical translation of the findings fromthese studies. Moreover, ischemic brain injuries, such as those causedby strokes, often elicit an inflammatory response that involvesactivation and migration of microglia and monocyte-derived macrophagesin the central nervous system. Within and adjacent to these injurysites, these immune cells adopt unique phenotypes with either protectiveor detrimental effects on neuron survival. Thus, there is a great needin the art for novel and effective treatments and protective compoundsfor neurological diseases and disorders, such as ischemic brain injuriescaused by stroke.

SUMMARY OF THE INVENTION

Various embodiments herein include a method of treating and/orameliorating the effects of a neurologic disease in a subject,comprising providing a composition comprising pertussis toxin (PTx), ora derivative, analog, pharmaceutical equivalent, and/or salt thereof,and treating and/or ameliorating the effects of the neurologic diseaseby administering a therapeutically effective dosage of the compositioncomprising pertussis toxin (PTx), or a derivative, analog,pharmaceutical equivalent, and/or salt thereof to the subject. Inanother embodiment, the neurologic disease is multiple sclerosis. Inanother embodiment, the neurologic disease is Systemic lupuserythematosus (SLE), Rheumatoid arthritis and Wegener's granulomatosis,complications related to the Human Immunodeficiency Virus (HIV),Guillain-Barre syndrome, meningitis, Alzheimer's disease, dementia, orParkinson's disease. In another embodiment, the subject is a human. Inanother embodiment, the subject is a mouse or rat. In anotherembodiment, the composition is administered intracerebroventricularly(icv) or intraperitoneally (ip). In another embodiment, ameliorating theeffects of the neurologic disease in the subject includes mitigatingclinical motor symptoms, minimizing T cell infiltration, and/orpreventing demyelination of the spinal cord. In another embodiment,administering the composition results in inhibition of migration ofmicroglia in the subject. In another embodiment, the composition isadministered to the subject in conjunction with G-protein, chemokineand/or adhesion blocking agents. In another embodiment, administeringthe composition results in increased vascular endothelial growth factor(VEGF) expression on neurons and/or increased angiogenesis. In anotherembodiment, administering the composition results in increased bloodvessel density in brain cortex and/or spinal gray matter. In anotherembodiment, the therapeutically effective dosage comprises at least 500ng PTx. In another embodiment, the therapeutically effective dosagecomprises at least 1000 ng PTx.

Other embodiments include a method of protecting against a neurologicdisease in a subject, comprising providing a composition comprisingpertussis toxin (PTx), or a derivative, analog, pharmaceuticalequivalent, and/or salt thereof, and protecting against the neurologicdisease by administering a therapeutically effective dosage of thecomposition comprising pertussis toxin (PTx), or a derivative, analog,pharmaceutical equivalent, and/or salt thereof to the subject. Inanother embodiment, the neurologic disease is multiple sclerosis. Inanother embodiment, the neurologic disease is a central nervous systemautoimmune disease. In another embodiment, the neurologic disease isSystemic lupus erythematosus (SLE), Rheumatoid arthritis and Wegener'sgranulomatosis, complications related to the Human ImmunodeficiencyVirus (HIV), Guillain-Barre syndrome, meningitis, Alzheimer's disease,dementia, or Parkinson's disease. In another embodiment, the subject isa human. In another embodiment, the subject is a mouse or rat. Inanother embodiment, the composition is administeredintracerebroventricularly (icv) or intraperitoneally (ip). In anotherembodiment, administering the composition results in inhibition ofmigration of microglia in the subject. In another embodiment,administering the composition results in increased vascular endothelialgrowth factor (VEGF) expression on neurons and/or increasedangiogenesis. In another embodiment, the therapeutically effectivedosage comprises at least 500 ng PTx. In another embodiment, thetherapeutically effective dosage comprises at least 1000 ng PTx.

Other embodiments include a pharmaceutical composition, comprising atherapeutically effective amount of pertussis toxin (PTx), or aderivative, analog, pharmaceutical equivalent, and/or salt thereof, anda pharmaceutically acceptable carrier. In another embodiment, thetherapeutically effective amount of PTx is about 1000 ng PTx.

Some embodiments include a method of reducing T cell infiltration into aneurological tissue in a human subject. For example, the method mayinclude providing a composition comprising pertussis toxin (PTx)comprising subunits A and B and administering (e.g.,intracerebroventricularly (icv) or intraperitoneally (ip)) atherapeutically effective dosage of the composition to the humansubject. In some aspects, the neurological tissue may comprise braintissue that has undergone an ischemic brain injury resulting from astroke (e.g., an ischemic stroke). In some embodiments, theadministration may also result in the inhibition of migration ofmicroglia in the human subject (e.g., migration to the site of injury).

In another embodiment, the composition is administered to the subject inconjunction with G-protein, chemokine and/or adhesion blocking agents.In another embodiment, administering the composition results inincreased vascular endothelial growth factor (VEGF) expression onneurons and/or increased angiogenesis. In another embodiment,administering the composition results in increased blood vessel densityin brain cortex and/or spinal gray matter. In another embodiment, thetherapeutically effective dosage comprises at least 500 ng PTx. Inanother embodiment, the therapeutically effective dosage comprises atleast 1000 ng PTx.

Some other embodiments may provide a method of protecting against anoccurrence of stroke (e.g., an ischemic stroke) in a human subject atrisk therefore. For example, the method may comprise the steps of:providing a composition comprising pertussis toxin (PTx) comprisingsubunits A and B and administering (e.g., intracerebroventricularly(icv) or intraperitoneally (ip)) a therapeutically effective dosage ofthe composition to the human subject. In some aspects, administration ofthe composition may result in inhibition of migration of microglia and Tcell infiltration to the site of the ischemic stroke.

Some embodiments may provide a method of reducing migration of microgliato a site of brain injury (e.g., caused by an ischemic stroke) in ahuman subject. For example, the method may comprise the steps ofproviding a composition comprising pertussis toxin (PTx) comprisingsubunits A and B and administering (e.g., intracerebroventricularly(icv) or intraperitoneally (ip)) a therapeutically effective dosage ofthe composition to the human subject. Moreover, in some embodiments,administration of the composition may also inhibit migration of T cellsto the site of the brain injury.

Other features and advantages of the invention will become apparent fromthe following detailed description, taken in conjunction with theaccompanying drawings, which illustrate, by way of example, variousembodiments of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Exemplary embodiments are illustrated in referenced figures. It isintended that the embodiments and figures disclosed herein are to beconsidered illustrative rather than restrictive.

FIG. 1 depicts EAE+PTx icv mice developed an attenuated and delayedcourse of EAE. Clinical scores were evaluated daily in EAE+PTx icv andcontrol mice and were plotted as the mean 6 S.D (n=12/group). Maximumclinical scores as well as scores on day 14 and 23 evidence markedattenuation of disease severity after PTx icv (P, 0.01). A dose responseto PTx icv is demonstrated as well. Mice receiving lower doses of PTxicv (400 ng and 200 ng) continued to manifest a dose dependent benefitcompared to the EAE controls (P, 0.05).

FIG. 2 depicts T cell proliferation responses to the Ag (MOG35-55peptide) were assessed in triplicate wells for each experiment. Itshowed a significant difference in PTx+EAE and EAE versus control (*p,0.01). But there was no difference between PTx+EAE and EAE mice. Resultsare expressed as Dcpm (mean cpm stimulated cultures−mean cpmunstimulated cultures). N=6/group.

FIG. 3 depicts flow cytometry analysis of mononuclear cells from thespleen on day 14. PTx icv does not alter the peripheral lymphocytesubpopulation in acute EAE. Dot plots of flow cytometry resultsgenerated after gating on lymphocytes (by forward and versus sidescatter) are shown for T (CD3+, CD4+, CD8+, CD4+/CD25+ and B(CD32/CD19+) cells. WT=wild type group. Absolute numbers of lymphocytesubpopulation, macrophage/microglia cells are shown in the followingtable. n=6/group. *p<0.05 compared with WT, **p<0.01 compared with WT.

FIG. 4 depicts attenuation of the progression of inflammation and tissueinjury in the CNS of mice that received PTx icv. Pathologicalexamination of spinal cord sections from EAE+PTx icv and EAE mice wereperformed at 7, 14, and 23 days post EAE induction to evaluate CNSinflammation, demyelination and axonal damage. In EAE+PTx icv mice, thenumber of immune-cell infiltrates (H&E staining, FIG. 4A-C) anddemyelination (Luxol fast blue staining, FIG. 4G) were bothsignificantly reduced at day 14 and 23 post EAE induction.Representative day 14 images of H&E staining (A-F) and LFB/PAS staining(G, H). B and C were inserts in A; E and F were inserts in D. Originalmagnification 640 in A, D, G and H; 6200 in B, C, E, F, and inserts in Gand H.

FIG. 5 depicts rabbit immunoglobulin G (IgG) penetration into thefrontal lobe parenchyma and thoracolumbar spinal cord in control, EAE,and EAE+PTx icv (n=7/group). Normal+IgG: age-controlled normal micewithout EAE receiving a single i.p. injection of rabbit IgG (100mg/mouse). 7 days EAE: EAE mice on day 7 post immunization; nopenetration of rabbit IgG observed in the brain or spinal cord. 14 daysEAE: EAE mice on day 14; marked penetration of rabbit IgG noted in bothbrain and spinal cord. 7 days EAE+PTx: EAE+PTx icv mice on day 7 postimmunization; marked penetration of the brain, but no penetration of thespinal cord. 14 days EAE+PTx: EAE+PTx icv mice on day 14 postimmunization, continued evidence of brain penetration, no penetration ofthe spinal cord. Note the dramatic opening of the BBB on Days 7 inEAE+PTx icv group relative to EAE on day 7.

FIG. 6 depicts western blot depicts measures of rabbit IgG. Lane 1:purified rabbit Ig G as the positive control; lane 2-7 correlates theplotted graph below. Statistical evaluation of optic density (OD)normalized to b-actin was obtained for each group. Mean 6 SD aredepicted (n=7 per group). *P<0.01, compared with normal control;**P<0.01, compared with normal control group and EAE.

FIG. 7 depicts inflammatory cytokines and cells in the spinal cord ofEAE and EAE+PTx icv mice (n=6/group). IL-17+/CD4+ cells were detected inthe meninges of the spinal cord in the EAE+PTx icv mice (A-C), whereasthese cells were diffusely identified in the spinal parenchyma in theEAE mice (D-F). Original magnification 6400. The western blot depictsmeasures of IL-17 (G), IL-6 (H) and TGF-b (I). In the spinal cord,elevated levels of all three were identified in the EAE mice relative tothe EAE+PTx icv mice. Statistical evaluation of optic density (OD)normalized to b-actin was obtained. Mean 6 SD are depicted (n=6 pergroup). *P<0.05, compared with normal control group; #P<0.05, comparedwith EAE group.

FIG. 8 depicts western blot measures of IL17 (A), IL6 (B) and TGF-b (C)in the brain of EAE+PTx icv compared with in EAE alone mice as well ascontrols. Statistical evaluation of optic density (OD) normalized tob-actin was obtained. Mean+/2 SD are depicted (n=6 per group). *P<0.05,compared with normal control group; #P<0.05, compared with EAE group.

FIG. 9 depicts anti-Iba1 immunostaining of spinal cord and brain of WT,EAE and EAE+PTx icv mice. Brain and spinal cord sections wereimmuno-stained at 7 days post MOG immunization with the anti-Iba1antibody. A: Low-magnification image of spinal cord section (Scalebar=200 mm). The anti-Iba1 antibody reacted strongly withamoeboid-shaped cells, corresponding to activated microglia in thespinal cord of EAE mice. This was significantly less prominent in theEAE+PTx icv mice. In WT controls, the antibody also effectively, butrather weakly, recognized ramified or resting microglia; these cellshave small bodies and finely branched processes. B: High-magnificationimage of the spinal cord sections (Scale bar=50 mm). C:Low-magnification image of cerebral cortex (Scale bar=200 mm). Theanti-Iba1 antibody reacted strongly with amoeboid shaped cells,corresponding to activated microglia in the brain of EAE+PTx icv mice.WT controls manifest ramified or resting microglia; whereas EAE micemanifest an intermediate stage. D: High-magnification image of the brainsections (Scale bar=50 mm). E-F. Microglia were quantified and comparedamong the groups by counting the number of cells in the field. Fiverandom fields at 40× fields were counted for each condition under adigital axoplan microscope. Results were shown as the cells counted per40× field. *p<0.05 compared with wt; **p<0.01, Compared with EAE.

FIG. 10 depicts PTx significantly reduced migration of stimulatedmicroglia. Microglia migration was quantified and compared among thegroups by counting the number of cells that migrated through themembrane to the lower chamber. Five random fields at 40× fields werecounted for each condition under a phase contrast microscope. Resultswere shown as the cells counted per 40× field (A and B). In PTx treatedgroups, cell migration was significantly reduced. *p<0.05 compared withPTx; **p<0.01, Compared with PTx+IFN, PTx and Control groups.

FIG. 11 depicts PTx ip (1000 ng) delayed the onset of motor symptoms anddecreased the severity of motor impairment (p<0.01) (FIG. 11). Theinventors evaluated whether A or B subunit alone was effective withequivalent dosage. Neither of them showed therapeutic effect. B subunitalone showed a trend in delaying the onset of motor deficits.

FIG. 12 depicts EAE+PTx ip mice exhibited markedly decreasedinfiltration of inflammatory cells in the spinal cord.

FIG. 13 depicts luxol fast blue staining which observed widespreaddemyelination zones in the white matter of the spinal cord of EAE micecompared to EAE+PTx ip mice.

FIG. 14 depicts PTx ip attenuates macrophage/microglia infiltration tothe CNS. In the brain and spinal cord of EAE mice, anti-Iba1 antibodyreacted strongly with amoeboid-shaped cells, corresponding to activatedmicroglia. Wild type controls manifest ramified or resting microglia;whereas EAE+PTx ip mice manifest intermediate responsiveness andramification.

FIG. 15 depicts PTx attenuated clinical deficits of EAE. Clinical scoreswere plotted as the mean±SD (n=12/group). Clinical signs began on day 13post-immunization and continued to worsen on day 19 in the EAE group. InPTx treatment group, no clinical sign was observed.

FIG. 16 depicts PTx attenuated inflammation in EAE. Pathologicalexamination of brain and spinal cord sections were performed at day 19post EAE induction to evaluate CNS inflammation. Abundant infiltratinginflammatory cells around blood vessels and in the parenchyma of brainand spinal cord were shown in the EAE group. They were significantlyreduced in the PTx treatment group. Representative images of H&Estaining were shown. Original magnification ×40; inserts ×200.

FIG. 17 depicts PTx attenuated demyelination in EAE. Pathologicalexamination of brain and spinal cord sections were performed at day 19post EAE induction to evaluate CNS demyelination. Massive subpialdemyelination with inflammatory cells infiltrating in parenchyma wereseen in both brain and spinal cord in EAE, especially in the spinalcord. They were significantly reduced in the PTx treatment group.Representative images of Luxol fast blue staining/PAS staining wereshown. Original magnification ×40; inserts ×200.

FIG. 18 depicts PTx increased VEGF expression. Immunohistochemistry ofbrain and spinal cord sections were performed at day 19 post EAEinduction. The expression of VEGF was increased significantly by PTxtreatment. The morphology of these cells is suggestive of neurons.Representative images of immunostaining were shown. Originalmagnification ×40; inserts ×200.

FIG. 19 depicts PTx increased VEGF expression on neurons. Doublestaining of brain and spinal cord sections with VEGF and NeuN antibodiesconfirmed VEGF expression on neurons. PTx significantly increased theexpression of VEGF on neurons. Original magnification ×200.

FIG. 20 depicts PTx increased angiogenesis. Brain and spinal cordsections were stained by Collagen IV to count the vessels. PTx treatmentincreased the vessel counts significantly versus EAE and control.Original magnification ×40; inserts ×200.

FIG. 21 depicts PTx increased protein levels of VEGF and Collagen IV inthe brain. Shown is the representative western blot depicting VEGF andCollagen IV from brain homogenate. Statistical evaluation of opticdensity (OD) normalized to β-actin was obtained. Both VEGF and collagentype IV were decreased in EAE (*P<0.05) and increased in the PTxtreatment group (**p<0.01).

FIG. 22 depicts PTx increased the expression of VEGF. Primary neuronswere cultured for 7 days and treated with PTx at the concentration of100 and 400 ng/ml for one day. Double staining with VEGF and Map2antibodies showed the expression of VEGF on PTx treated primary neurons.The VEGF was increased in a dose-dependent patent (*P<0.01). Originalmagnification ×200.

FIG. 23 depicts a correlation of cerebral blood flow (CBF) and infarctvolume. (a) Shown is the region of interest for CBF (upper panel) andinfracted areas (lower panel). (b) Representative CBF and T2 weightedmagnetic resonance imaging images showing that CBF determines theinfarct volume. (c1) the correlation between absolute CBF (aCBF) andinfarct volume in three slices. (c2) the correlation between relativeCBF (rCBF) and infarct volume in three slices. (d1) the correlationbetween infarct volume in three slices and all slices. (d2) thecorrelation between rCBF and infarct volume in all slices. Scale bar=1mm.

FIG. 24 depicts the fact that PTx treatment attenuated infarct volumeafter permanent middle cerebral artery occlusion. (a) Infarct volume wasexponentially dependent on relative cerebral blood flow (rCBF) in bothcontrol (blue line, the same data as FIG. 1d 2) and PTx treated mice(red line). Notice that the exponential model remained valid inpredicting infarct volume based on rCBF after PTx treatment. (b) WhenrCBF was between 0.4 and 0.6 (the middle CBF group), PTx treatmentattenuated infarct volume (n=5, *p<0.01); PTx treatment did notattenuate the infarct volume when rCBF was lower than 0.4 (the low CBFgroup); or higher than 0.6 (the high CBF group, virtually no infarctionwere observed). (c) absolute CBF (aCBF) was measured in both ipsilateraland contralateral hemispheres, no significant difference was seen incontrol and PTx treated mice when they were divided into 3 groupsaccording to the rCBF level.

FIG. 25 depicts the fact that PTx treatment protected neurons againstglutamate in vitro. (a) Representative images of live neurons after3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)treatment in four groups, glutamate damaged most neurons and PTxprotected neurons against this damage. (b) MTT assay was performed toevaluate the neurons survival after glutamate treatment. PTx treatmentreduced the neuronal death caused by glutamate. (c) lactatedehydrogenase (LDH) release from neurons was calculated in all groups.Glutamate increased the LDH release, while PTx treatment attenuated theincrease of LDH (n=6, *p<0.01). Representative images were shown, scalebars=50 μm.

FIG. 26 depicts the fact that PTx treatment reduced calcium influx inneurons. (a) Calcium influx in neurons was recorded. Calcium influx inneurons occurred soon after glutamate treatment and reached the peakfast, while PTx treatment decreased the influx dramatically. (b) Foldincrease of calcium was calculated, which represented the volume ofcalcium influx. Calcium was increased more than five folds in thecontrol group, while less than three folds were seen in the PTxtreatment group. (c) Time to reach the half of the peak calciumconcentration was recorded, which represented the speed of calciuminflux. PTx treatment decreased the speed of calcium influxsignificantly (n=20, *p<0.01).

FIG. 27 depicts the fact that PTx treatment decreased the caspase-3positive cells after permanent middle cerebral artery occlusion. (a)Staining with caspase-3 antibody in the ischemic area 24 h after pMCAO,(b) semi-quantification analysis of the number of caspase-3 positivecells showing PTx treatment decreased the apoptosis (n=4, *p<0.01), (c)Invert images with Image J software to measure the density of caspase-3positive cells, (d) semi-quantification analysis of the density ofcaspase-3 positive cells showing PTx treatment decreased the caspase-3density (n=4, *p<0.01). Representative images were shown, scale bars=50μm.

FIG. 28 depicts that CX3CR1 deficiency attenuates infarct volume andneurological deficit after middle cerebral artery occlusion. (A) Infarctvolume was assessed by T2-weighted images at 24 and 72 hourspost-ischemia. (B) Quantification of T2 images shows that the infarctvolume was attenuated by CX3CR1 deficiency 72 hours after middlecerebral artery occlusion (MCAO). P<0.0001 for genotype, P=0.0194 fortime point, and P=0.0011 for interaction by two-way analysis ofvariance. **P<0.01 by Bonferroni post-hoc tests. (C) Clinical assessmentdemonstrated that CX3CR1^(−/−) mice have better neurological deficitscores than wild-type (WT) mice 72 hours after MCAO. P=0.0049 forgenotype, P=0.0262 for time point, and P=0.0062 for interaction bytwo-way analysis of variance. *P<0.05 by Bonferroni post-hoc tests. n=6per group.

FIG. 29 depicts that CX3CR1 deficiency attenuates neuronal apoptosisafter middle cerebral artery occlusion. (A) Double staining with cleavedCaspase 3 (red) and NeuN (green) antibodies in peri-infarct area ofwild-type (WT) and CX3CR1^(−/−) mice 72 hours after middle cerebralartery occlusion. (B) Quantification of Cleaved-Caspase 3/NeuN positivecells. CX3CR1^(−/−) mice have fewer Cleaved-Caspase 3⁺NeuN⁺ cells in theperi-infarct zone. **P<0.01 by Student's t-test. n=4 per group. Scalebars=50 μm.

FIG. 30 depicts that CX3CR1 deficiency attenuates infiltration and leadsto a different M1/M2 polarization pattern on microglia/macrophages inipsilateral hemisphere of middle cerebral artery occlusion mice. (A)Staining with anti-Iba-1 antibody in the ipsilateral hippocampus,striatum, cortex and peri-infarct zone of CX3CR1^(−/−) and wild-type(WT) mice 72 hours after middle cerebral artery occlusion (MCAO). (B)Quantification of Iba-1 positive cells. CX3CR1^(−/−) mice have fewerIba-1 positive cells (macrophages/microglia) in the ipsilateralhippocampus, striatum, cortex and peri-infarct zone, while nodifferences are observed in these sites of the contralateral hemisphere.P<0.0001 by two-way analysis of variance for genotype, localization andinteraction. *P<0.05, **P<0.01 by Bonferroni post-hoc tests. n=4 pergroup. Scale bars=50 μm. (C) Flow cytometry analysis of CD45^(hi)/CD11b⁺and CD45^(low)/CD11b⁺ cells isolated from the ipsilateral andcontralateral hemispheres of CX3CR1^(−/−) and WT mice 72 hours afterMCAO by gating on Ly6G⁻ events (Ly6G vs FSC). (D) Quantification of thenumber of events in the CD45^(hi)/CD11b⁺/Ly6G⁻ (left) andCD45^(low)/CD11b⁺/Ly6G⁻ (right) gate. Statistical analysis showsCX3CR1^(−/−) mice have fewer CD45^(hi)/CD11b⁺/Ly6G⁻ as well asCD45^(low)/CD11b⁺/Ly6G⁻ cells in the ipsilateral hemisphere.CD45¹¹¹/CD11b⁺/Ly6G⁻: P=0.0001 for genotype, P<0.0001 for localizationand interaction by two-way analysis of variance.CD45^(low)/CD11b⁺/Ly6G⁻: P=0.0112 for genotype, P<0.0001 forlocalization, and P=0.0152 for interaction by two-way analysis ofvariance. **P<0.01 by Bonferroni post-hoc tests. n=4 per group. (E)Microglia/macrophage in ischemic hemisphere of CX3CR1^(−/−) brainpolarize toward the M2 phenotype. Real-time reverse-transcriptionpolymerase chain reaction was performed using total RNA extracted fromsorted CD45⁺/CD11b⁺/Ly6G⁻ microglia/macrophage at 72 hours after MCAO.Data are expressed as fold change vs sham-operated controls. **P<0.01with Student's t-test for Yml, Mcrl, and iNOS, respectively. n=4 pergroup.

FIG. 31 depicts that suppressed proliferation of CD45^(low)/CD11b⁺/Ly6G⁻and CD45^(hi)/CD11b⁺/Ly6G⁻ cells in the ipsilateral hemisphere of CX3CR1mice after middle cerebral artery occlusion. (A) Flow cytometry analysisof proliferation of Ly6G⁻ gated CD45^(low)/CD11b⁺ (microglia) andCD45^(hi)/CD11b⁺ (macrophages/activated microglia) population with5-bromo-2-deoxyuridine (BrdU) incorporation in the ipsilateralhemisphere of wild-type (WT) and CX3CR1^(−/−) mice 72 hours after middlecerebral artery occlusion (MCAO), compared to their contralateral(unlesioned) hemisphere controls. CD45^(hi)/CD11b⁺/Ly6G⁻ cellproliferation was not shown in the contralateral due to their very lowto undetectable presence. (B) Graph presents quantification ofmicroglia/macrophage proliferation measured by flow cytometry. Dataindicate a marked reduction in CD45^(low)/CD11b⁺/Ly6G⁻ cellproliferation and a modest but significant reduction inCD45^(hi)/CD11b⁺/Ly6G⁻ cell proliferation in the ipsilateral hemisphereof CX3CR1^(−/−) mice compared to WT mice 72 hours after MCAO. P<0.0001for genotype and localization, P=0.0017 for interaction by two-wayanalysis of variance. *P<0.05, **P<0.01 by Bonferroni post-hoc tests.n=4 per group.

FIG. 32 depicts that CX3CR1 deficiency attenuates reactive oxygenspecies generation in brain after middle cerebral artery occlusion. (A)Reactive oxygen species (ROS) were evaluated by Xenogen IVIS200 imagerin wild-type (WT) and CX3CR1^(−/−) mice in vivo. (B) Quantification ofROS. No difference in ROS levels were observed between the two groups 24hours after MCAO. ROS level decreased in CX3CR1^(−/−) mice 72 hoursafter MCAO compared to 24 hours (P<0.0001 for genotype and time point,P=0.0036 for interaction by two-way analysis of variance; **P<0.01 byBonferroni post-hoc tests) and is significantly less in CX3CR1^(−/−)mice relative to WT mice (*P<0.05 by Bonferroni post-hoc tests). n=6 pergroup. p/s/cm²/sr, photons per second per centimeter squared persteradian. (C) Immunohistochemistry for 4-hydroxy-2-nonenal (4-HNE) and8-hydroxy-2-deoxyguanosine (8-OHdG) in the ischemic lesion 24 and 72hours after MCAO. (D) Reduction of the number of stained cells in theCX3CR1^(−/−) mice compared with WT mice. P<0.0001 for genotype, P=0.0003for oxidative marker, and P=0.9827 for interaction by two-way analysisof variance. **P<0.01 by Bonferroni post-hoc tests. n=5 per group scalebars: 50 μm.

FIG. 33 depicts that CX3CR1 deficiency impairs inflammatory signaling inmicroglia and macrophage in ischemic brain. (A) The amounts of IL-1β,IL-6, and TNF-α in brain homogenates from wild-type (WT) andCX3CR1^(−/−) mice 72 hours after middle cerebral artery occlusion (MCAO)were measured with ELISA. IL-1β: P=0.0018 for genotype, P=0.0002 forlocalization, and P=0.0052 for interaction by two-way analysis ofvariance; IL-6: P=0.0010 for genotype, P<0.0001 for localization, andP=0.0058 for interaction by two-way analysis of variance; TNF-α:P<0.0001 for genotype, P=0.0240 for localization, and P=0.0063 forinteraction by two-way analysis of variance. *P<0.05, **P<0.01 byBonferroni post-hoc tests. n=4 per group. (B) IL-1β, IL-6, and TNF-αexpression were analyzed by flow cytometry within the CD11b⁺Ly6G⁻ gate.Representative histograms show IL-1β, IL-6, and TNF-α expression in thecontralateral (blue) and ipsilateral (red) hemispheres of CX3CR1^(−/−)and WT mice at 72 hours after MCAO. Mean fluorescent intensity isindicated within each representative histogram. (C) The number of IL-1β,IL-6, and TNF-α-producing CD11b⁺Ly6G⁻ cells was quantified from ischemicbrain of CX3CR1^(−/−) and WT mice at 72 hours after MCAO withflowcytometry. IL-1β⁺/CD11b⁺/Ly6G⁻: P=0.0030 for genotype, P=0.0002 forlocalization, and P=0.0052 for interaction by two-way analysis ofvariance; IL-6⁺/CD11b⁺/Ly6G⁻: P=0.0030 for genotype, P=0.0002 forlocalization, and P=0.0052 for interaction by two-way analysis ofvariance; TNF-α⁺/CD11b⁺/Ly6G⁻: P=0.0386 for genotype, P=0.0332 forlocalization, and P=0.0173 for interaction by two-way analysis ofvariance. *P<0.05 by Bonferroni post-hoc tests. n=4 per group.

DETAILED DESCRIPTION OF THE INVENTION

All references cited herein are incorporated by reference in theirentirety as though fully set forth. Unless defined otherwise, technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Singleton et al., Dictionary of Microbiology and MolecularBiology 3rd ed., J. Wiley & Sons (New York, N.Y. 2001); March, AdvancedOrganic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley& Sons (New York, N.Y. 2001); and Sambrook and Russel, MolecularCloning: A Laboratory Manual 3rd ed., Cold Spring Harbor LaboratoryPress (Cold Spring Harbor, N.Y. 2001), provide one skilled in the artwith a general guide to many of the terms used in the presentapplication.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. Indeed, the present invention is inno way limited to the methods and materials described.

As disclosed herein, the inventors investigated the effects of Pertussistoxin (PTx) administered intracerebroventricularly (icv) as well asintraperitoneally (ip) in preventing downstream immune cell infiltrationand demyelination of the spinal cord. EAE was induced in C57BL/6 micewith MOG35-55. PTx icv at seven days post MOG immunization resulted inmitigation of clinical motor symptoms, minimal T cell infiltration, andthe marked absence of axonal loss and demyelination of the spinal cord.Integrity of the blood brain barrier was compromised in the brainwhereas spinal cord BBB integrity remained intact. PTx icv markedlyincreased microglia numbers in the brain preventing their migration tothe spinal cord. An in vitro transwell study demonstrated that PTxinhibited migration of microglia. Centrally administered PTx abrogatedmigration of microglia in EAE mice, limiting the inflammatory cytokinemilieu to the brain and prevented dissemination of demyelination.

As further disclosed herein, the inventors have provided evidence of theusage of PTx as a therapeutic agent to treat autoimmune disease, andprovide insight into the etiological mechanism of autoimmune diseasesand provide a therapeutic model demonstrating attenuation of the lesionand clinical manifestations of EAE with early administration of icv andip PTx. Understanding the mechanism of PTx allows implementation ofother directed methods to mimic the effects of PTx therapeuticallyutilizing G protein, chemokine, and adhesion blocking agents. Inaddition, the design and development of effective treatment strategiessurrounding the unique concept of translocated inflammation providefurther insight into the mechanism of therapies. By applying PTx, theinventors successfully attenuate the motor deficits in EAE. Beyond itsimplications for multiple sclerosis, the understanding of microglia andT cell translocation in central nervous system (CNS) and themanipulation of its regulation have a broad impact in other autoimmunediseases such as Systemic lupus erythematosus (SLE), Rheumatoidarthritis and Wegener's granulomatosis as well as infectious disordersaffecting the immune system such as HIV, Guillain-Barre syndrome andmeningitis. Finally, in light of the role of immune modulation in theeffective treatment of neurodegenerative diseases, there is also animpact on neurodegenerative diseases such as Alzheimer's and Parkinson'sdisease and the continuum between immunologic disease andneurodegeneration.

As further disclosed herein, the inventors have provided evidence of theusage of PTx to treat and or prevent one or more insults or injuries tothe neurological system. For example, in some embodiments, the treatmentand/or prevention of the one or more insults or injuries to theneurological system may be provided via administering a therapeuticallyeffective dosage of a composition comprising pertussis toxin (PTx),including subunits A and B, or a derivative, analog, pharmaceuticalequivalent, and/or salt, thereof to the subject. In some embodiments,the one or more insults or injuries to the neurological system maycomprise an ischemic brain injury, such as a brain injury caused by astroke. As such, administration of PTx may prevent or treat ischemicbrain injuries arising from an ischemic stroke in a human subject. Forexample, prior to or after the occurrence of an ischemic brain injury,administration of PTx may reduce and/or eliminate one or more signs ofan inflammatory response.

In some embodiments, PTx may be administered to individuals at risk fora first ischemic brain injury or may have already suffered a firstischemic brain injury and may be at risk for subsequent ischemic braininjuries. In some aspects, an individual/subject who has not previouslysuffered an ischemic brain injury, such an as ischemic stroke, may bedetermined by a healthcare provider to be a risk for experiencing anischemic brain injury (e.g., via an evaluation of current state ofhealth, personal health history, family health history, environmentalfactors, genetic factors, etc.). As such, according to some embodiments,a therapeutically effective amount of a composition comprising PTx canbe administered to the subject at risk for the stroke to prevent theoccurrence of the stroke. In other aspects, the subject may have alreadysuffered from one or more ischemic brain injuries, such as a stroke,such that the subject may be at an increased risk of addition strokes.In such circumstances, the composition comprising a therapeuticallyeffective amount of PTx may also be administered to the subject at riskto reduce the risk of, and protect against the occurrence of a stroke inthe subject.

In some aspects, administration of PTx may reduce infiltration/migrationof one or more types of leukocytes, such as T cells and microglia to thesite of the ischemic injury, such as the brain or immediately adjacenttissues. As such, the potentially harmful inflammatory response may beblunted to reduce the likelihood of the occurrence of the ischemic braininjury or treat the resulting injury if administered after occurrence.

In one embodiment, the present invention provides a method of treating adisease or disorder in a subject by administering a therapeuticallyeffective dosage of a composition comprising pertussis toxin (PTx), or aderivative, analog, pharmaceutical equivalent, and/or salt, thereof tothe subject. In another embodiment, the present invention provides amethod of mitigating the effects of and/or slowing progression of thedisease by administering a therapeutically effective dosage of acomposition comprising pertussis toxin (PTx), or a derivative, analog,pharmaceutical equivalent, and/or salt, thereof to the subject. Inanother embodiment, the administration of the composition preventsand/or reduces the likelihood of susceptibility to developing thedisease relative to a healthy individual. In another embodiment, thesubject is a mouse. In another embodiment, the subject is a human. Inanother embodiment, the disease is a neurodegenerative disease,including for example, Alzheimer's disease, dementia, and Parkinson'sdisease. In one embodiment, the disease or disorder is an ischemic braininjury, such as an ischemic injury caused by a stroke. In anotherembodiment, the disease is an autoimmune disease, including but notlimited to acute demyelinating encephalomyeliltis, multiple sclerosis,and systemic lupus erythematosus, Rheumatoid arthritis, and Wegener'sgranulomatosis. In another embodiment, administering the compositionresults in inhibition of migration of microglia (e.g., to the site ofthe ischemic injury caused by a stroke). In another embodiment,administering the composition results in increased vascular endothelialgrowth factor (VEGF) expression on neurons and/or increasedangiogenesis. In another embodiment, the therapeutically effectivedosage comprises at least 1000 ng PTx. In another embodiment, thedisease is an infectious disorder affecting the immune system such asbut not limited to HIV, Guillain-Barre syndrome and meningitis. Inanother embodiment, the invention may provide treatment for inflammatoryspinal cord injury. In another embodiment, the treatment may beadministered intracerebroventricularly (icv) and/or intraperitoneally(ip).

In another embodiment, the present invention provides a compositioncomprising a therapeutically effective dosage of pertussis toxin (PTx),or a pharmaceutical equivalent, analog, derivative, and/or salt thereof,and a pharmaceutically acceptable carrier. In another embodiment, thetherapeutically effective dosage is about 1000 ng PTx.

In some embodiments of the invention, the effective amount of pertussistoxin (PTx), or a pharmaceutical equivalent, analog, salt, and/orderivative in the composition can be in the range of about 10-100 ng,100-200 ng, 200-300 ng, 300-400 ng, 400-500 ng, 500-600 ng, 600-700 ng,700-800 ng, 800-900 ng, 900-1000 ng, 1000-1100 ng, 1100-1200 ng,1200-1300 ng, 1300-1400 ng, 1400-1500 ng, 1500-1600 ng, 1600-1700 ng,1700-1800 ng, 1800-1900 ng, 1900-2000 ng, 2000-2100 ng, 2100-2200 ng,2200-2300 ng, 2300-2400 ng, 2400-2500 ng, 2500-2600 ng, 2600-2700 ng,2700-2800 ng, 2800-2900 ng, or 2900-3000 ng.

In some embodiments of the invention, the effective amount of pertussistoxin (PTx), or a pharmaceutical equivalent, analog, salt, and/orderivative in the composition can be in the range of about 10-100ng/day, 100-200 ng/day, 200-300 ng/day, 300-400 ng/day, 400-500 ng/day,500-600 ng/day, 600-700 ng/day, 700-800 ng/day, 800-900 ng/day, 900-1000ng/day, 1000-1100 ng/day, 1100-1200 ng/day, 1200-1300 ng/day, 1300-1400ng/day, 1400-1500 ng/day, 1500-1600 ng/day, 1600-1700 ng/day, 1700-1800ng/day, 1800-1900 ng/day, 1900-2000 ng/day, 2000-2100 ng/day, 2100-2200ng/day, 2200-2300 ng/day, 2300-2400 ng/day, 2400-2500 ng/day, 2500-2600ng/day, 2600-2700 ng/day, 2700-2800 ng/day, 2800-2900 ng/day, or2900-3000 ng/day.

In some embodiments of the invention, the effective amount of pertussistoxin (PTx), or a pharmaceutical equivalent, analog, salt, and/orderivative in the composition can be in the range of about 10-100 mg,100-200 mg, 200-300 mg, 300-400 mg, 400-500 mg, 500-600 mg, 600-700 mg,700-800 mg, 800-900 mg, 900-1000 mg, 1000-1100 mg, 1100-1200 mg,1200-1300 mg, 1300-1400 mg, 1400-1500 mg, 1500-1600 mg, 1600-1700 mg,1700-1800 mg, 1800-1900 mg, 1900-2000 mg, 2000-2100 mg, 2100-2200 mg,2200-2300 mg, 2300-2400 mg, 2400-2500 mg, 2500-2600 mg, 2600-2700 mg,2700-2800 mg, 2800-2900 mg, or 2900-3000 mg.

Typical dosages of an effective amount of pertussis toxin (PTx), or apharmaceutical equivalent, analog, salt, and/or derivative can be in theranges recommended by the manufacturer where known therapeutic compoundsare used, and also as indicated to the skilled artisan by the in vitroresponses or responses in animal models. For example, one of skill inthe art may readily calculate and prepare the equivalent dosages of PTxfor human patients based on an effective dosage of 1000 ng/dayadministered to a mouse model. The same or similar dosing can be used inaccordance with various embodiments of the present invention, or analternate dosage may be used in connection with alternate embodiments ofthe invention, such as administrating in conjunction with or withoutvarious G-proteins, chemokines and/or adhesion blocking agents. Theactual dosage can depend upon the judgment of the physician, thecondition of the patient, and the effectiveness of the therapeuticmethod based, for example, on the in vitro responsiveness of relevantcultured cells or histocultured tissue sample, or the responses observedin the appropriate animal models.

As readily apparent to one of skill in the art, various embodimentsdescribed herein may be used to treat any number of conditions anddiseases that affect the central nervous system, motor deficits, spinalcord injury and/or inflammation, and demyelination, and the invention isnot in any way limited to only treatment of multiple sclerosis.Similarly, as described herein, the inventors have determined thatpertussis toxin may be distinguished from other possible therapeuticlesions by mediating therapeutic effects immunologically, as opposed tobeing neurotransmitter driven. Thus, as readily apparent to one of skillin the art, any number of additional compositions or substitutes mayalso act through a similar mechanism, including pharmaceuticalequivalents, derivatives, analogs, and/or salts, or other compounds andagents that mimic pertussis toxin's therapeutic utilization of Gprotein, chemokine and adhesion blocking, and the invention is notlimited only to pertussis toxin itself.

EXAMPLES

The following examples are provided to better illustrate the claimedinvention and are not to be interpreted as limiting the scope of theinvention. To the extent that specific materials are mentioned, it ismerely for purposes of illustration and is not intended to limit theinvention. One skilled in the art may develop equivalent means orreactants without the exercise of inventive capacity and withoutdeparting from the scope of the invention.

Example 1 Generally

Experimental autoimmune encephalomyelitis (EAE) models are importantvehicles for studying the effect of infectious elements such asPertussis toxin (PTx) on disease processes related to acutedemyelinating encephalomyelitis (ADEM) or multiple sclerosis (MS). PTxhas pleotropic effects on the immune system. The inventors investigatedthe effects of PTx administered intracerebroventricularly (icv) inpreventing downstream immune cell infiltration and demyelination of thespinal cord.

Methods and Findings: EAE was induced in C57BL/6 mice with MOG35-55. PTxicv at seven days post MOG immunization resulted in mitigation ofclinical motor symptoms, minimal T cell infiltration, and the markedabsence of axonal loss and demyelination of the spinal cord. Integrityof the blood brain barrier was compromised in the brain whereas spinalcord BBB integrity remained intact. PTx icv markedly increased microglianumbers in the brain preventing their migration to the spinal cord. Anin vitro transwell study demonstrated that PTx inhibited migration ofmicroglia.

Centrally administered PTx abrogated migration of microglia in EAE mice,limiting the inflammatory cytokine milieu to the brain and preventeddissemination of demyelination. The effects of PTx icv warrants furtherinvestigation and provides an attractive template for further studyregarding the pleotropic effects of infectious elements such as PTx inthe pathogenesis of autoimmune disorders.

Example 2 EAE Induction and Treatment

The animals were kept in groups on a 12:12 h light/dark cycle with foodand water ad libitum. EAE was induced in female C57BL/6 mice (6-8 weeksold, Taconic Laboratory, New York, USA) by subcutaneous injection with200 mg myelin oligodendrocyte glycoprotein (MOG35-55;M-E-V-G-W-Y-R-S-P-F-S-R-V-V-H-L-Y-R-N-G-K, Bio-synthesis Inc.Lewisville, Tex.), dissolved in an emulsion of 50 ml of completeFreund's adjuvant containing 0.5 mg of heat killed Mycobacteriumtuberculosis (CFA, Difco Laboratories, Detroit, Mich.) and 50 ml ofphosphate buffered saline (PBS). On the day of immunization (day 0) and48 h later (day 2), PTx (List Biological laboratories Inc.) 200 ng inPBS was injected into the mouse tail vein. Neurological functional testswere performed by an examiner blinded to the treatment status of eachanimal. Functional data were collected on 7 mouse groups (n=12/group), 3PTx icv treatment groups (EAE+PTx icv 1000 ng, 400 ng, and 200 ng), 2EAE groups (EAE and EAE+normal saline (NS) icv) and 2 non-EAE controlgroups (normal+1000 ng PTX icv and CFA+1000 ng PTX icv). Neurologicalassessments were reported using a five-point standardized rating scaleto evaluate motor deficit: 0 no deficit; 1 tail paralysis; 2 unilateralhind limb weakness; 3 incomplete bilateral hind limb paralysis and/orpartial forelimb weakness; 4 complete hind limb paralysis and partialforelimb weakness; 5 moribund state or death. Scores were measured dailyfor 23 days. The onset of disease was calculated by determining thetotal number of days from MOG35-55 immunization to the onset of symptomsin individual animals. Maximal motor scores and motor scores at day 14and 21 were compared as were onset of disease.

Example 3 Stereotactic Intracerebroventricular Injection

Mice were anaesthetized by injection of a ketamine/xylamine cocktail onday 7 after MOG35-55 immunization and mounted in a stereotactic device.A fine hole was drilled through the skull giving access to the surfaceof the brain 0.7 mm caudal to bregma and 1.0 mm lateral to the sagittalsuture. A guarded, 27-gauge 0.5-in needle was stereotactically inserted,targeting the lateral ventricle (3.5 mm depth). A 10.0-ml Hamilton 1700series gastight syringe was used to inject 2 ml of normal saline, or PTx(500 mg/ml dissolved in normal saline) into the lateral ventricle over afive-minute period.

Example 4 Immunohistochemistry

Mice were euthanized at day 7 14 or day 23 post immunization. Terminallyanesthetized mice were intracardiacally perfused with saline followed by4% paraformaldehyde. The spinal cord and brain were embedded in paraffinand cut into serial 6-mm thick coronal slides. Histological evaluationwas performed by staining with hematoxylin and eosin (H&E), Luxol fastblue/periodic acid Schiff agent (LFB/PAS), and Bielschowsky silverimpregnation to assess inflammation, demyelination, and axonalpathology, respectively. Histological scores assessing the degree ofinflammation, demyelination, and axonal loss in the spinal cord wereevaluated using a semi-quantitative system. In brief, the degree ofinflammation was assessed by counting the number of cellular infiltratesin the spinal cord. Digital images were collected using an Axoplanmicroscope (Zeiss, Thornwood, N.Y.) under bright field setting using a40× objective. Severity of inflammatory cell infiltration on H&Estaining was scored using the following scale: 0, no inflammation; 1,cellular infiltrates only around blood vessel and meninges; 2, mildcellular infiltrates in parenchyma (1-10/section); 3, moderate cellularinfiltrates in parenchyma (11-100/section); and 4, serious cellularinfiltrates in parenchyma (0.100/section). Serial sections ofparaformaldehyde-fixed spinal cord were stained with Luxol fast blue formyelin and were assessed in a blinded fashion for demyelination usingthe following scale: 0, normal white matter; 1, rare foci; 2, a fewareas of demyelination; 3, confluent perivascular or subpialdemyelination; 4, massive perivascular and subpial demyelinationinvolving one half of the spinal cord with presence of cellularinfiltrates in the CNS parenchyma; and 5, extensive perivascular andsubpial demyelination involving the whole cord section with presence ofcellular infiltrates in the CNS parenchyma. Axonal loss was assessedusing the following scale: 0, no axonal loss; 1, a few foci ofsuperficial axonal loss which involves less than 25% of the lateralcolumns; 2, foci of deep axonal loss and that encompasses over 25% ofthe lateral columns; and 3, diffuse and widespread axonal loss. At leastsix serial sections of each spinal cord from each mouse were scored andstatistically analyzed by ANOVA. Data were presented as Mean 6 Standarddeviation (SD). Immunohistochemistry was performed with rabbitpolyclonal antibodies against IL-6 (1:2000, #ab6672, Abcam Inc;Cambridge, Mass.), and TGF-b (1:3000, #ab66043, Abcam Inc; Cambridge,Mass.) to identify crucial pro-inflammatory cytokines; and againstionized calcium binding adaptor molecule 1 (Iba-1, 1:2500, WakoChemicals Inc. LA) for microglia and glia fibrillary acidic protein(GFAP, 1:400, Millipore Corporation, Billerica, Mass.) for astrocytes.Sections of brain and spinal cord stained with anti-Iba1 allowedquantification of microglia and assessment of its morphology. Theinventors performed a morphological analysis of the changes observed andquantified the microglia in sections of cerebral cortex and spinal cord.Th17 cells were identified by double immunostaining for CD4 (1:1600,Chemicon, Temecula, Calif.), and IL-17 (1:3000, rabbit mAb, #ab40663,Abcam Inc., Cambridge, Mass.) with two fluorescent conjugated secondaryantibodies (FITC conjugated and Texas Red conjugated). Immunolabelingwas detected by applying the peroxidase-antiperoxidase procedure with 3,39-diaminobenzidine (DAB) as cosubstrate. Negative control slidesreceived identical preparations for immunostaining, except that primaryantibodies were omitted.

Example 5 Western Blot Protein Analysis

Aliquots of equal amount of proteins were loaded onto a 10%SDS-polyacrylamide gel. After gel electrophoresis, blots weresubsequently probed with primary antibodies (anti-IL-6, 1:1000 #ab6672,anti-IL-17, 1:3000 #ab40663, anti-TGF-b 1:1000 #ab66043 Abcam Inc;Cambridge, Mass.). For detection, horseradish peroxidase-conjugatedsecondary anti-rabbit antibody was used (1:10,000, #7074, Cell signalingtechnology; Danvers, Mass.), followed by enhanced chemiluminescencedevelopment (ECL kit, #34077, Thermo Scientific Pierce, Rockford Ill.).Normalization of results was ensured by running parallel Western blotswith b-actin antibody (1:25,000 #ab49900, Abcam Inc; Cambridge, Mass.).The optical density was quantified using an image densitometer (ModelGS-670, BioRad, Hercules, Calif.). The data are presented as apercentage of target protein relative to b-actin. A value of p, 0.05 isconsidered significant.

Example 6 BBB Studies

Qualitative (immunohistochemistry) and quantitative (Western blot)analyses of exogenous rabbit IgG penetration across the BBB into the CNSwere used to evaluate the extent of regional breakdown of the BBB in EAEand EAE+PTx icv mice [20]. Normal and PTx icv (without EAE) were used ascontrols. Mice were injected intraperitoneally (i.p.) with 100 mgpurified rabbit IgG (Ir-Rb-Gf, Innovative research, Novi, Mich., USA) onday 7 (four hours after PTx icv in the EAE+PTx icv group) or day 14.Animals were euthanized 18-19 hours after the injection. Forimmunohistochemistry, paraffin embedded sections were probed directlywith biotinylated anti-rabbit IgG (1:100; Vector laboratories,Brulingame, Calif.). For Western blot, the horseradishperoxidase-labeled anti-rabbit antibody (1:5000, Cell SignalingTechnology, Davers, Mass.) was used.

Example 7 T Cell Proliferation Assays

Animals were sacrificed on day 14. Mononuclear cells were isolated fromthe spleen and were suspended in culture medium containing DMEMsupplemented with 1% penicillin-streptomycin and 10% (v/v) FBS(Invitrogen Life Technologies). Mononuclear cells were then seeded onto96-well plates at a concentration of 46105 cells/well. Ten microlitersof MOG35-55 peptide (10 mg/ml), PLP139-151 peptide (10 mg/ml), or Con A(5 mg/ml; Sigma-Aldrich) were then added in triplicate into the wells.After 3 days of incubation, the cells were pulsed for 18 h with 10-mlaliquots containing 1 mCi of [methyl-3H] thymidine (42 Ci/mmol; AmershamBiosciences). Cells were harvested onto glass fiber filters, and thethymidine incorporation was measured. The results were expressed as Dcpm(DCPM) (mean cpm stimulated cultures−mean cpm unstimulated cultures).

Example 8 Flow Cytometry Analysis

To evaluate the frequency of CD4+, CD8+, CD4+/CD25+, CD32/CD19+,CD45+/CD11b+ cells, spleen mononuclear cell culture was prepared fromeach group on day 14 (the peak of autoimmune response). Single cellsuspensions (26106 cells/5 ml BD tube) were incubated with combinationsof fluorescent antibodies, for 30 min at 4 uC: CD3 (17A2), CD19 (1D3),CD4 (GK 1.5), CD8 (53-6.7), CD25 PC61.5), CD11b (M1/70), and CD45(RA3-6B2). The indicated antibodies were fluorescently tagged witheither FITC, PE, allophycocyanin, PE-Cy5, PE-Cy7 or APC-Cy7. Allpurchased from BD Pharmingen. After incubation, each suspension waswashed twice (400 g, 5 min, 4 uC) with PBS containing 2% bovine serumalbumin (BSA) and was resuspended in PBS with 0.5% of paraformaldehyde.Appropriate isotype controls were included. All samples were analyzed onAccuri C6 Flow Cytometer (Accuri Cytometers Inc, USA). Data wereanalyzed on CFlow Plus software. The number of mononuclear cells permouse spleen was counted on hemocytometer and the absolute number of acell subset was calculated based on the percentage of cells stained forthe appropriate markers.

Example 9 Cytokine Quantification by Enzyme-Linked Immunosorbent Assay(ELISA)

To assess cytokine expression, spleen mononuclear cells were prepared asdescribed above. Suspensions were incubated in RPMI-1640 medium at 37 uCfor 2 days (26106 cells/well) with or without antigens (MOG35-55 10mg/ml or Con A 5 mg/ml, Sigma, USA). Supernatants were collected andaliquoted in 96-well plate precoated with antibodies to Interferon c(IFN-c), Tumor Necrosis Factor a (TNF-a), Interleukin-2 (IL-2),Interleukin-4 (IL-4), Interleukin-6 (IL-6) and Interleukin-10 (IL-10)(ELISA Max™ Set Deluxe, BioLegend Inc. San Diego, Calif.). Opticaldensity was measured at 450 nm on Model 680 Microplate Reader (Bio-RadLaboratories, Corston, UK). The optical density was quantified byGraphPad Prism 4 (GraphPad Software, Inc) using the standard curveprovided by the manufacturer.

Example 10 Primary Microglia Cell Culture

Cortical tissue was harvested from 0 or 1-day-old C57/BL6 mouse pups(Taconic, Hudson, N.Y.). Meninges and visible vasculature were removedunder a dissecting microscope. Cortical tissue was digested in theDMEM/F12 media (Invitrogen Corporation, CA) containing 0.25% trypsin andEDTA (1 mM) at 37 uC for 15 minutes. The digested tissue was resuspendedin 20 ml media containing DMEM/F12 supplemented with 15% heatinactivated fetal bovine serum, 5% Horse serum (Sigma, St. Louis, Mo.)and 1% Penicillin-Streptomycin and filtered through a 70-mm nylon mesh(BD Biosciences, San Jose, Calif.). The cells were washed and seeded ina 75 cm2 flask in fresh culture medium (3-4 Pups/per flask). The purityof the microglia cultures was assessed by double-immunostaining withmicroglial special markers anti Ionized calcium binding adaptor molecule1 (Iba-1, 1:2500, Wako Chemicals Inc. LA) and glia fibrillary acidicprotein (GFAP, 1:400, Millipore Corporation, Billerica, Mass.). Thepurity of this primary microglia cell culture is about 90-95%.

Example 11 Microglia Migration Assay

The migration of microglia in vitro was determined by using Transwell(pore size 8-mm, Corning, VWR, San Dimas, Calif.). Cellfree DMEM/F-12media (0.8 ml) with or without IFN-c (20 ng/ml, BD Biosciences, SanJose, Calif.) was placed in the lower chamber. Microglia suspension (0.1ml, 56104 cell/per well) was placed in upper chamber and incubated withor without PTx (100 ng/ml, Campbell, Calif.) for 24 hours at 37 uC. Theinserts were then removed and the upper surface was carefully cleansedwith cotton pads. Cells on the lower surface were air dried and stainedfor microglia. Microglial migration was quantified and compared amongthe groups by counting the number of cells that migrated through themembrane to the lower chamber. Five random fields at 40× fields werecounted for each condition under a phase contrast microscope. Eachexperiment was repeated three times. Results were shown as the cellscounted per 40× field.

Example 12 Statistical Analysis

Data were analyzed with SPSS version 10 for windows. The two wayanalysis of variance was applied to determine the significance of thedifference among the experimental groups. Kruskal-Wallis nonparametricanalysis was used for data presented as percentage. The Mann-Whitney Utest was used when Kruskal-Wallis showed significance among groups. P,0.05 was considered significant.

Example 13 Results—Table 1

TABLE 1 Splenocytes from EAE and EAE + PTx mice expressed elevatedlevels of TNF-α, IFN-γ, IL-2, IL-6, and IL-4 compared to WT controls.Pg/ml WT EAE EAE + PTx TNF-α 3.0 +/− 0.9 46.7 +/− 2.0* 49.3 +/− 1.9*IFN-γ 9.9 +/− 8.9 2385.9 +/− 556.9* 2636.2 +/− 186.9* IL-2 5.4 +/− 0.6105.9 +/− 26.0* 138.2 +/− 23.1* IL-6 14.1+/−3.8  1144.0 +/− 211.5*1047.0 +/− 186.1* IL-4 1.3 +/− 0.4 170.9 +/− 62.5* 144.3 +/− 11.3* Therewas no significant differences in cytokine production in EAE and EAE +PTx. *P, 0.001, compared with WT. Abbrevation: WT: wild type, EAE:experimental autoimmune encephalomyelitis model group, EAE + PTx: EAEmice with cerebral ventricle injection of Pertussis toxin (PTx).

Example 14 Results—Table 2

TABLE 2 Histopathological analyses of inflammatory parameters,demyelination and axonal damage in the spinal cord of C57BL/6 mice at 7,14, and 23 days after MOG₃₅₋₅₅ EAE induction. EAE EAE + PTx icv P valueInflammation (H and E) day 7 0.25 +/− 0.27 0.08 +/− 0.20 0.260 Day 143.33 +/− 0.75 1.33 +/− 0.75 0.001* Day 23 3.42 +/− 0.58 1.33 +/− 0.68<0.001* Demyelination (Fast blue) Day 7 0.25 +/− 0.27 0.08 +/− 0.200.260 Day 14 3.66 +/− 0.98 0.83 +/− 0.98 0.001* Day 23 3.75 +/− 0.931.16 +/− 0.98 0.001* Axonal loss (silver staining) Day 7 0.83 +/− 0.200.04 +/− 0.10 0.664 Day 14 2.42 +/− 0.86 0.66 +/− 0.51 0.002* Day 232.58 +/− 0.97 0.58 +/− 0.49 0.001* Data presented as Mean +/− SD

Example 15 PTx Icv Prevents Against Dissemination of Motor Deficits inEAE and has a Dose Effect

PTx icv (1000 ng) delayed the onset of motor symptoms (11.660.64 versus8.560.75, p, 0.05) and decreased the severity of motor impairment(maximal clinical score 0.3560.07 vs. 3.2560.37, p, 0.01) (FIG. 1). Theinventors evaluated whether there was a dose effect associated withadministration of PTx icv (200 ng, 400 ng, and 1000 ng). There was asignificant dose effect. The 1000 ng group provided a significantlygreater therapeutic response than the 400 ng, and the 400 ng greaterthan the 200 ng (p, 0.05) which also provided a significant therapeuticresponse relative to EAE (p, 0.05) (FIG. 1). To control for potentialeffects of icv administration, EAE mice were treated with same volume ofnormal saline icv (EAE+NS icv). Motor deficits were unchanged comparedto EAE alone (FIG. 1). To determine whether the effects of the spinalcord lesion could be alleviated following symptom onset, PTx icv wasadministered immediately after the onset of measurable motor deficits(clinical score. 0.5; day 9+ post MOG35-55 inoculation). The delayedadministration did not alter the clinical course of EAE (n=6).

Example 16 The Variation in Clinical Disease is not Due to Differencesin Auto-Reactive T Cell Priming

To investigate whether an enhanced expansion of auto-reactive T cellscould be responsible for the observed clinical differences in EAE versusEAE+PTX icv, T cells were re-challenged with MOG35-55 in vitro. Nodifferences were observed between EAE and EAE+PTX icv regarding thecapacity of T cells to proliferate in response to recall antigen (FIG.2). Furthermore there was no difference in T cell subpopulations (CD4+,CD8+, CD4+/CD25+), B cell (CD32/CD19+), and macrophage/microglia(CD45+/CD11b+) (FIG. 3). Nor is there a pattern shift in Th1/Th2 betweenthe two groups (Table 1).

Example 17 PTx Icv Attenuates Spinal Cord Leukocyte Infiltration andDemyelination in EAE

On day 14 and 23, H&E staining in the cross-sectional of the spinal cordof EAE mice showed widespread infiltration of inflammatory cells in thespinal cord (FIG. 4D-F). In contrast, EAE+PTx icv mice exhibitedmarkedly decreased infiltration of inflammatory cells in the spinal cordon day 14 and 23. (FIG. 4A-C, Table 2). To determine the degree ofdemyelination, we stained sections of spinal cord with Luxol fast blueand observed widespread demyelination zones in the white matter of thespinal cord of EAE mice on day 14 and 23 (FIG. 4H). In contrast, on day14 and 23, mice that received PTx icv had minimal evidence ofdemyelination indicated by a markedly attenuated course of disease (FIG.4G, Table 2). Marked axonal loss characterizes the MOG35-55 model ofEAE, and this is evident in the spinal sections of the EAE mice assessedwith Bielschowsky silver impregnation. Attenuation of axonal injury isevidenced in EAE+PTx icv mice (Table 2).

Example 18 PTx Icv Increases BBB Permeability in EAE

The inventors determined BBB integrity by localizing rabbit IgG in theCNS in EAE and EAE+PTx icv before (day 7) and during the peak (day 14)of symptomatic disease. On day 7, rabbit IgG immunoreactivity wasobserved in the brains of EAE+PTx icv but not in EAE mice (FIG. 5, 6).In the spinal cord no immunoreactivity was observed in either group. Onday 14, EAE mice demonstrated immunoreactivity diffusely throughout theparenchyma of the spinal cord with minimal evidence of reactivity in thebrain. EAE+PTx icv mice showed rabbit IgG immunoreactivity in the brain,but not in the spinal cord (FIG. 5, 6). To control for potential effectsof PTx on BBB integrity, separate from its exacerbation of EAE relatedinflammation, mice were treated with 1000 ng PTx icv but were notexposed to MOG35-55. In contrast to EAE+PTx icv mice (FIG. 5, 6), micethat received only PTx icv exhibited no accumulation of rabbit IgG inthe brain or the spinal cord. Thus, the BBB breakdown described abovewas caused by the effect of PTx icv in the context of EAE.

Example 19 PTx Icv Preferentially Induces the Development ofMyelin-Reactive Th-17 Cells in the Brain

T helper cell lineage development depends on local cytokine milieus andspecific immune factors. Emerging evidence supports the pathonogmonicrole of Th-17 cells in EAE and the role of PTx in the induction of Th-17[28]. For the Th-17 cells, TGF-b and IL-6 drive the initial lineagecommitment. The inventors quantified the Th-17 cell concentration in ourmodel after PTx icv was administered. In the spinal cord, the presenceof IL-17 CD4 cells was rare and limited to the meninges in the EAE+PTxicv mice (FIG. 7A-C), whereas a considerable number of Th-17 cells wereidentified in the spinal parenchyma of the EAE mice (FIG. 7D-F). Theprotein levels of IL-17, IL6 and TGF-b (FIG. 7G-I) were significantlyelevated in the spinal cord of the EAE relative to the EAE+PTx icv mice(p, 0.05), correlating the spinal cord pathology in EAE mice.

In the brain, the EAE+PTX icv mice exhibited infiltrating leucocyteswhich stained positive for CD 4 and IL-17. The majority of thesecolocalized cells were in the periventricular white matter, confirmingthe infiltration of proinflammatory of Th-17 cells induced by PTx icv.Whereas, in the EAE alone mice, the presence of Th-17 cells in the brainwas limited to the meninges. The protein level of IL-17, IL-6, and TGF-bwere significantly elevated in the brain of EAE+PTx icv mice, relativeto the controls and the EAE alone mice (FIG. 8) (p, 0.05). In normalcontrol and CFA+PTx icv groups, no IL-17+ cells were detected in brain.

Example 20 PTx Icv Retains Macrophage/Microglia and to a Lesser Degree TCell Infiltration to the Brain Preventing Dissemination to the SpinalCord

The most salient finding of PTx icv on day 7 post immunization was theparenchymal infiltration of macrophage/microphage (Iba1), and to alesser magnitude T cell (CD4), in the brain (FIG. 9). In the brain ofEAE+PTx icv mice, anti-Iba1 antibody reacted strongly withamoeboid-shaped cells, corresponding to activated microglia on day 7.Wild type controls manifest ramified or resting microglia; whereas EAEmice manifest intermediate responsiveness and ramification (FIG. 9-C, D,F). In contrast, the spinal cord of EAE mice showed amoeboid-shapedcells that stained strongly with anti-Iba1 antibody, corresponding toactivated microglia (FIG. 9-A, B, E). To further determine the effect ofPTx on microglia migration, the inventors utilized the Transwell toassess in vitro migration. PTx significantly inhibited the migration ofmicroglia with and without IFN-c stimulation (FIG. 10).

Example 21 Therapeutic Effect of PTx

PTx results in evidence of: 1) dose and time course dependentattenuation of motor clinical symptoms; 2) In the spinal cord, typicallythe most affected region in the traditional EAE model, evidence ofminimal T cell infiltration, and the marked absence of axonal loss anddemyelination; 3) abrogation of the migration of microglia as well as Tcells to the lesion target and 4) modulation blood brain barrier (BBB)integrity. These results indicate that PTx icv/ip results in atherapeutic response in the EAE model.

The data demonstrates that neurodegenerative changes in the spinal cordare directly impacted by the therapeutic effects of PTx. PTx isrecognized as an immunoadjuvent and has been used to increase diseaseseverity; however, in this case it has a therapeutic effect,demonstrating the therapeutic effect of PTx given in a single dose onEAE. Successful demonstration of the mechanism of its dichotomous effectalso provide a clearer understanding of its role in autoimmune diseases.Secondly, the inventors demonstrate the concept of a therapeutic lesion.Though the concept of a therapeutic lesion, with actual placement of alesion in humans, has been seen in the neurodegenerative disorder, suchas Parkinson's disease, in that situation the mechanism is thought to beneurotransmitter driven. Data described herein has shown that PTxadministered through the ventricle as well as ip results in atherapeutic brain lesion which is mediated immunologically i.e.increased adhesion molecule activity and local infiltration butdecreased migrational activity in RAE. and the results support this.

PTx icv:

1. PTx icv prevents against dissemination of motor deficits in EAE andhas a dose effect.

2. The variation in clinical disease is not due to differences inauto-reactive T cell priming.

3. PTx icv attenuates spinal cord leukocyte infiltration anddemyelination in EAE

4. PTx icv increases BBB permeability in EAE

5. PTx icv preferentially induces the development of myelin-reactiveTh-17 cells in the brain.

6. PTx icv retains macrophage/microglia and to a lesser degree T cellinfiltration to the brain preventing dissemination to the spinal cord.

PTx ip:

1. PTx ip has similar effects on motor deficits in EAE: PTx ip (1000 ng)delayed the onset of motor symptoms and decreased the severity of motorimpairment (p<0.01) (FIG. 11). The inventors evaluated whether A or Bsubunit alone was effective with equivalent dosage. Neither of themshowed therapeutic effect. B subunit alone showed a trend in delayingthe onset of motor deficits, but it was not significant (FIG. 11).

2. PTx ip attenuates spinal cord leukocyte infiltration anddemyelination in EAE: H&E staining in the cross-sectional of the spinalcord of EAE mice showed widespread infiltration of inflammatory cells inthe spinal cord. By contrast, EAE+PTx ip mice exhibited markedlydecreased infiltration of inflammatory cells in the spinal cord (FIG.12). This is consistent with Luxol fast blue staining which observedwidespread demyelination zones in the white matter of the spinal cord ofEAE mice compared to EAE+PTx ip mice (FIG. 13).

3. PTx ip attenuates macrophage/microglia infiltration to the CNS: Inthe brain and spinal cord of EAE mice, anti-Iba1 antibody reactedstrongly with amoeboid-shaped cells, corresponding to activatedmicroglia. Wild type controls manifest ramified or resting microglia;whereas EAE+PTx ip mice manifest intermediate responsiveness andramification (FIG. 14).

Example 22 VEGF and Angiogenesis—Generally

Vascular endothelial growth factor (VEGF) and angiogenesis play animportant role in the pathophysiology of experimental autoimmuneencephalomyelitis (EAE) and multiple sclerosis (MS). The inventorsinvestigated whether PTx can increase VEGF expression and angiogenesiswhich in turn lead to beneficial effects in EAE model. EAE was inducedas by MOG 35-55 in C57BL/6 mice. Clinical scores of EAE were evaluateddaily for 19 days. Brain and spinal cord samples were stained byhematoxylin and eosin (H&E), Luxol fast blue/periodic acid Schiff agent(LFB/PAS) and immunohistochemistry for VEGF, NeuN and Collagen IV.Western blot protein analysis was used to assess the protein levels ofVEGF and collagen IV. In vitro study on primary neuronal culture wasdone to assess the effect of PTx on VEGF expression on neurons. Theinventors found that the treatment of PTx attenuates inflammation anddemyelination and therefore the clinical deficits in EAE. PTx increasesVEGF expression and angiogenesis in vivo and in vitro. The findingssuggest that upregulation of endogenous VEGF on neurons plays aprotective role in EAE and it is a potential target in treatment formultiple sclerosis.

Example 23 VEGF and Angiogenesis—Animals and EAE Induction

All experimental procedures were approved by the Institutional AnimalCare and Use Committee of the Barrow Neurological Institute andperformed according to the Revised Guide for the Care and Use ofLaboratory Animals. The animals were kept in groups on a 12:12 hlight/dark cycle with food and water ad libitum.

EAE was induced. Briefly, female C57BL/6 mice (6-8 weeks old, TaconicLaboratory, New York, USA) were subcutaneously injected with 200 μgmyelin oligodendrocyte glycoprotein (MOG35-55;M-E-V-G-W-Y-R-S-P-F-S-R-V-V-H-L-Y-R-N-G-K, Bio-synthesis Inc.Lewisville, Tex.), dissolved in an emulsion of 50 μl of completeFreund's adjuvant containing 0.5 mg of heat killed Mycobacteriumtuberculosis (CFA, Difco Laboratories, Detroit, Mich.) and 50 μl ofphosphate buffered saline (PBS). On the day of immunization (day 0) and48 h later (day 2), PTx (List Biological laboratories Inc.) 200 ng inPBS was injected intraperitoneally (ip). An additional 1000 ng PTx wasadministered ip on day 7 in the PTx treatment group.

Neurological functional tests were performed by an examiner blinded tothe treatment status of each animal. Clinical grades of EAE wereassessed using a five-point standardized rating scale: 0=no deficit;1=tail paralysis; 2=unilateral hind limb weakness; 3=incompletebilateral hind limb paralysis and/or partial forelimb weakness;4=complete hind limb paralysis and partial forelimb weakness; 5=moribundstate or death. Functional data were collected on 3 mouse groups(n=12/group): normal control group, EAE group and PTx treatment group.Scores were recorded daily.

Example 24 VEGF and Angiogenesis—Immunohistochemistry

Mice were euthanized at day 19 post immunization. Terminallyanesthetized mice were perfused intracardiacally with saline followed by4% paraformaldehyde. The fixed spinal cord and brain were embedded inparaffin and cut into serial 6 μm thick coronal slides. Histologicalevaluation was performed by staining with hematoxylin and eosin (H&E),Luxol fast blue/periodic acid Schiff agent (LFB/PAS) to assessinflammation and demyelination respectively.

Histological scores assessing the degree of inflammation anddemyelination in the spinal cord and brain were evaluated using asemi-quantitative system. In brief, the degree of inflammation wasassessed by counting the number of cellular infiltrates in the spinalcord. Digital images were collected using an Axoplan microscope (Zeiss,Thornwood, N.Y.) under bright field setting using a 40× objective.Severity of inflammatory cell infiltration on H&E staining was scoredusing the following scale as described (Okuda et al., 1999): 0=noinflammation; 1=cellular infiltrates only around blood vessel andmeninges; 2=mild cellular infiltrates in parenchyma (1-10/section);3=moderate cellular infiltrates in parenchyma (11-100/section); and4=severe cellular infiltrates in parenchyma (>100/section).

Serial sections of paraformaldehyde-fixed spinal cord and brain werestained with Luxol fast blue for myelin and were assessed in a blindedfashion for demyelination using the following scale: 0=normal whitematter; 1=rare foci; 2=a few areas of demyelination; 3=confluentperivascular or subpial demyelination; 4=massive perivascular andsubpial demyelination involving one half of the spinal cord or brainwith presence of cellular infiltrates in the CNS parenchyma; and5=extensive perivascular and subpial demyelination involving the wholecord section or brain with presence of cellular infiltrates in the CNSparenchyma. At least six serial sections of each spinal cord from eachmouse were scored and statistically analyzed by ANOVA. Data werepresented as Mean±Standard deviation (SD).

Immunohistochemistry was performed with antibodies against VEGF(NG1651636, Millipore Corporation, Billerica, Mass.) to identifypro-angiogenesis factors; and against Nestin (LV1634942, MilliporeCorporation, Billerica, Mass.) and Collagen IV (ab19808, Abcam Inc.,Cambridge, Mass.) to identify the density of blood vessels.Immunolabeling was detected by applying the peroxidase-antiperoxidaseprocedure with 3, 3′-diaminobenzidine (DAB) as cosubstrate.

For double fluorescent staining, antibodies against NeuN (MAB377,Millipore Corporation, Billerica, Mass.) and VEGF (NG1651636, MilliporeCorporation, Billerica, Mass.) were used to identify the expression ofVEGF on neurons. The sections were incubated in 5% FBS in PBST for 1hour, and then incubated in the mixture of two primary antibodies for 1hour at room temperature, followed by incubation with two fluorescentconjugated secondary antibodies (FITC conjugated and Texas Redconjugated) in PBST for 30 min at room temperature. Adjacent sectionswere used to detect co-localization. Respective negative controls thatomit primary antibodies and positive controls were applied for eachcase.

Example 25 VEGF and Angiogenesis—Western Blot Protein Analysis

Aliquots of equal amount of proteins were loaded onto an 8%SDS-polyacrylamide gel. After gel electrophoresis, blots weresubsequently probed with primary antibodies (VEGF, collagen IV). Fordetection, horseradish peroxidase-conjugated secondary antibody was used(7074, Cell signaling technology; Danvers, Mass.), followed by enhancedchemiluminescence development (ECL kit, 34077, Thermo Scientific Pierce,Rockford Ill.).

Normalization of results was ensured by running parallel Western blotswith β-actin antibody (sc-47778, Santa Cruz Biotechnology, Inc., SantaCruz, Calif.). The optical density was quantified using an imagedensitometer (Model GS-670, BioRad, Hercules, Calif.). The data arepresented as a percentage of target protein relative to β-actin. A valueof p<0.05 is considered significant.

Example 26 VEGF and Angiogenesis—In Vitro

To prepare primary neuronal culture, cells were collected from cerebralcortices of 0 or 1-day-old C57/BL6 mouse pups (Taconic, Hudson, N.Y.).Pups were dipped in 95% ethanol inside a cell culture hood. The wholebrains were exposed and the meninges were removed under a dissectingmicroscope. Both sides of cortex were removed and put into dish withNeurobasal Medium (Invitrogen Corporation, CA). The cortices were cutand minced mechanically. Tissues were incubated in Papain digestionsolution (Worthington, Biochemical, Lakewood) at 37° C. for 20 minuteswith continuous shaking (150 rpm). Digestion was stopped by addition of10% FBS (Sigma, St. Louis, Mo.) and filtered through a 70 um cellstrainer. It was centrifuged for 3 min (1500 rpm) and the supernatantwas discarded. 2 ml Neurobasal Medium supplemented with 0.5% L-glutamineand 2% B27 serum-free supplement (Invitrogen Corporation, CA) was addedto re-suspend the cells in a flask. Cells were plated into Poly-D-lysinecovered dishes. They were cultured in 5% CO2 atmosphere at 37° C. Mediumwas replaced every 3-4 days.

On day 7, primary neuronal cells were treated with PTx at theconcentration of 100 ng/ml or 400 ng/ml. 24 hours after treatment, thecells were fixed with 4% paraformaldehyde. Cells were double stainedwith antibodies VEGF (NG1651636, Millipore Corporation, Billerica,Mass.) and Map2 (3-1103, Gainesville, Fla.) to measure the expression ofVEGF on neurons. The expression of PTx on neurons was evaluated bycalculating the mean density with the VisionWorks LS Image Acquisitionand Analysis Software.

Example 27 VEGF and Angiogenesis—the Treatment of PTx AttenuatedClinical Deficits of EAE

To investigate whether the treatment of PTx alleviated clinical deficitsof EAE, clinical scores were evaluated daily in each group (control, EAEand PTx). After MOG induction, motor symptoms were observed on day 13and continued to worsen up to day 19 in the EAE group. In the PTxtreatment group, no clinical signs were observed during the same period.

Example 28 VEGF and Angiogenesis—the Treatment of PTx AttenuatedInflammation and Demyelination in EAE

At day 19 after immunization, mice were sacrificed to detected theinflammation and demyelination by H&E and Luxol fast blue staining.Infiltrating inflammatory cells were abundant around blood vessels andin the parenchyma of brain and spinal cord in the EAE group. In the PTxtreatment group, the number of inflammatory cells were markedly reduced(FIG. 2). Massive perivascular and subpial demyelination withinflammatory cells infiltrating the parenchyma were seen especially inthe spinal cord in the EAE group. In the PTx treatment group, few fociof demyelination were observed. Semi-quantitative analysis showed therewas a significant difference in the degree of inflammation anddemyelination in the brain and spinal cord between EAE and PTx treatmentgroups (Table 3).

Example 29 VEGF and Angiogenesis—PTx Increased VEGF Expression andAngiogenesis

Sections were stained with antibodies of VEGF and collagen IV to detectchange of angiogenesis in different groups. Expression of VEGF on thecells located in brain cortex and spinal gray matter was increasedsignificantly in the PTx treatment group. Double staining with VEGF andNeuN antibodies confirmed these cells were neurons. Consistently, bloodvessel counts by collagen IV staining were increased significantly inthe PTx treatment group. In the inflammatory cell infiltrating sites anddemyelination lesion areas in the EAE group, the expression of VEGF andblood vessel counts were increased, but the overall protein levels ofVEGF and collagen type IV by Western blot were decreased (p<0.05).

Example 30 VEGF and Angiogenesis—PTx Increased the Expression of VEGF InVitro

To further delineate the effect of PTx on VEGF expression in vitro, theinventors cultured primary neurons. On day 7, neuronal cells weretreated with PTx at the concentration of 100 and 400 ng/ml. Theexpression of VEGF after 24-hour treatment was significantly increasedand this increase was dose-dependent (p<0.01).

Example 31 VEGF and Angiogenesis—Table 3

Table 3 depicts semi-quantification analysis of inflammation anddemyelination in the brain and spinal cord at 19 days afterimmunization.

TABLE 3 EAE EAE + PTx P value Inflammation (H and E) Brain 3.4 +/− 0.551.2 +/− 0.45 0.0001 Spinal cord 3.6 +/− 0.55 1.4 +/− 0.55 0.0002Demyelination (Fast Blue) Brain 3.6 +/− 0.55 1.0 +/− 0.71 0.0002 Spinalcord 4.2 +/− 0.84 1.4 +/− 0.55 0.0002

Example 32 VEGF and Angiogenesis

As described herein, the inventors demonstrated that PTx treatmentincreases VEGF expression and angiogenesis. They have also shown thatthe increase of VEGF is from neurons and blood vessel density isincreased in brain cortex and spinal gray matter. In vitro study hasfurther established the dose-dependent effect of PTx on VEGF expression.Importantly, the inventors found that angiogenesis plays a protectiverole in EAE and that improving angiogenesis is one of the mechanisms ofPTx preventing central nervous system autoimmune disease in the EAEmodel.

VEGF and angiogenesis play a role in EAE. Although the inventors foundan upregulation of VEGF and vessel counts in the lesion areas in EAE,this transient increase is likely local and reactive to inflammation. Itdoesn't alert the overall decrease in VEGF and angiogenesis in EAE. Thiswould explain the seemingly contradictory results in previous studies.

Neuronal VEGF plays a protective role in most CNS diseases. Studiesdemonstrated that VEGF has neuroprotective effects and can stimulateneuron outgrowth and survival. Neuron degeneration in motor systemdiseases has been linked to down regulation of endogenous VEGF, such asamyotrophic lateral sclerosis (ALS) and Kennedy disease. It also hasbeen demonstrated that down regulated VEGF by genetic manipulationresults in degeneration of motor neurons. Interestingly, it has beenpreviously reported that within the spinal cord in the course ofautoimmune encephalomyelitis not only myelin but also neurons aresubject to lymphocyte attack and may degenerate. Loss of neurons hasbeen demonstrated in EAE. The inventors found the expression of VEGF onneurons was up regulated significantly after PTx treatment, andadministration of PTx prevented the inflammation and demyelination inEAE. This supports that up regulation of neuronal VEGF play a protectiverole in EAE.

In summary, the inventors have shown that administration of PTxattenuates the inflammation and demyelination in EAE through upregulating endogenous VEGF on neurons and angiogenesis. The findingssupport that endogenous VEGF on neurons plays a protective role in EAEand it is a potential target in treatment for multiple sclerosis.

Example 33 Materials and Methods for Examples 34-38

All methodologies contained herein are from Z. Tang et al., Pertussistoxin reduces calcium influx to protect ischemic stroke in a middlecerebral artery occlusion model, J. of Neurochemistry 2015, vol. 135,998-1006. This reference is incorporated by reference in its entiretyfor all purposes.

C57BL/6 mice were purchased from Taconic (Oxnard, Calif., USA). Allanimals were housed in pathogen free conditions at the animal facilitiesof the Barrow Neurological Institute. All experimental procedures wereapproved by the Institutional Animal Care and Use Committee of theBarrow Neurological Institute and performed according to the RevisedGuide for the Care and Use of Laboratory Animals.

Thirty (30) C57BL/6 male mice (age 10-14 weeks) were randomly dividedinto either a PTx-treatment group or a control group, i.e. 15 mice ineach group. The average weight was 25.3±1.4 g in the PTx-treatment groupand 24.4±1.8 g in the control group. Randomization was based onalternating selection of mice whoever came the next upon arrival. Peoplewho performed the surgery, immunohistochemistry and magnetic resonanceimaging (MRI) were blinded to the randomization.

Mice were under Ketamine/Xylazine anesthesia (80 mg/kg Ketamine and 10mg/kg Xylazine, injected intraperitoneal, i.p.). Standard asepticsurgical procedures were used throughout the procedure. The animals werekept warm with a carefully monitored heating lamp to maintain bodytemperature at 37° C. during surgery and recovery after surgery.Following the induction of anesthesia, the surgical site was cleaned,and the hair was shaved with an electric razor. The exposed skin wasthen disinfected. Ophthalmic ointment was applied to the eyes to preventdrying during the procedure. A 6-0 surgical nylon monofilament with arounded tip was introduced into the right internal carotid arterythrough the external carotid stump. It was then advanced 10-11 mm pastthe carotid bifurcation until a slight resistance was felt. At thispoint, the intraluminal filament blocked the origin of the middlecerebral artery. After the permanent middle cerebral artery occlusion(pMCAO) procedure, mice were monitored carefully to assure temperatureregulation while recovering from anesthesia. Mice was housed one percage for recovery. Food, water, and hydrating gel were made available onthe floor of the cage. Mice were monitored daily for any signs ofinfection, and antibiotics would be administered as needed. In ourexperience, surgical site infections were uncommon. Analgesics would beadministered for any signs of distress, as per animal care facilityguidelines.

MRI was performed on a 7T small animal MRI, 30-cm horizontal-boremagnet, and a BioSpec Avance III spectrometer (Bruker Daltonics Inc.,Fremont, Calif., USA) with a 116-mm high power gradient set (600 mT/m)and a 72-mm whole-body mouse transmit/surface coil configuration.Isoflurane anesthesia was induced and maintained for each animal at 3%and 1.5% respectively. It was delivered with medical air at 1.5 L/min.During MRI scan, the animal's respiration was continually monitored by asmall animal monitoring and gating system (SA Instruments, Stoney Brook,N.Y., USA) via a pillow sensor positioned under the abdomen. Mice wereplaced on a heated circulating water blanket (Bruker, Billerica, Mass.,USA) to maintain body temperature at 36-37° C. T2-weighted images wereacquired 24 h after pMCAO to assess the infarct volume, using a RapidAcquisition with Refocused Echoes sequence with parameters: TR=3000 ms,effective TE=60 ms, Rapid Acquisition with Refocused Echoes factor=8. 20slices were acquired with thickness=0.5 mm, field of view 2.56×2.56 cm,matrix 128×128, total scan time 3 min 12 s. In order to assess thecerebral blood flow (CBF) in the MCA territory, images were acquired 24h after pMCAO, using a Continuous Arterial Spin Labeling sequence withparameters: TR=3000 ms, TE=6.95 ms, segments=4, slice thickness=1.5 mm,field of view 2.0×2.0 cm, matrix 64×64, total scan time 20 min. MRI datawere analyzed using the MEDx3.4.3 software package (Medical Numerics,Germantown, Md., USA) on a LINUX workstation.

Mice were euthanized with isoflurane anesthesia at 24 h after inducingpMCAO. They were then perfused intracardiacally with saline followed by4% paraformaldehyde for a total of 30 min. The fixed brain was embeddedin paraffin and cut into serial 6 μm thick coronal slides.Immunohistochemistry was performed with antibodies against caspase-3(#9662S; Cell Signaling Technology, Beverly, Mass., USA). Immunolabelingwas detected by applying the peroxidase-antiperoxidase procedure with3,3′-diaminobenzidine as a co-substrate. Respective negative controlsthat omit primary antibodies and positive controls were applied for eachcase. Digital images were collected by using a microscope (Bx53; OlympusAmerica Inc., Center Valley, Pa., USA) under the bright field setting.The number and the density of positively stained cells were counted at40× magnification in matched sections. Results were presented aspositive cells per mm², with areas measured from 40× images using ImageJ. 1.34vi software (National Institutes of Health, Bethesda, Md., USA).

To prepare primary neuronal culture, cells were collected from cerebralcortices of 0 or 1-day-old C57/BL6 mouse pups (Taconic, Hudson, N.Y.,USA). Pups were dipped in 95% ethanol inside a cell culture hood at23-25° C. The meninges were removed under a dissecting microscope on topof ice. The cortices were cut and minced mechanically in the NeurobasalMedium (#21103-049; Life technologies, Grand Island, N.Y., USA). Tissueswere incubated in Papain digestion solution (#LS003124; WorthingtonBiochemical Corporation, Lakewood, N.J., USA) at 37° C. for 20 min withcontinuous shaking (150 rpm, ThermoScientic MaxQ5000 shaker, Waltham,Mass., USA). Digestion was stopped by adding 10% Fetal Bovine Serum(#f2442; Sigma-Aldrich Co, St Louis, Mo., USA). The solution wasfiltered through a 70 μm cell strainer. It was centrifuged for 3 min(250 g) to remove the supernatant at 23-25° C. Re-suspended cells wereplaced into Poly-D-lysine covered dishes in the Neurobasal Mediumsupplemented with 0.5% L-glutamine and 2% B27 serum-free supplement(#17504-001; Life Technologies). They were cultured in the 5% CO₂atmosphere at 37° C. Medium was replaced every 3-4 days.

Glutamate excitotoxicity was measured using the lactate dehydrogenase(LDH)-based CytoTox96-non-radioactive cytotoxicity assay kit (TOX7;Sigma-Aldrich Co) in accordance with the manufacturer's protocol. Theabsorbance was measure at 490 nm. Each assay was tested in triplicate.Percentage of specific lysis was determined as: (Experimentalrelease−target spontaneous release)/(Target maximum release−targetspontaneous release)×100. The maximum release was determined bydetecting the absorbance of the target cells lysed with LDH assay lysissolution. Viability of glutamate-treated neurons was tested by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)assay. Data were expressed as percentage of viable cells compared withthe control culture.

On day 10, primary neurons on coverslips were washed in warmed Hank'sbalanced salt solution (HBSS). They were then incubated in a 35 mm dishcontaining 2 mL Fluo-3/AM work solution (3 μM Fluo-3/AM in HBSS) for 20min at 37° C. Coverslips were washed again in HBSS and placed on thestage of laser scanning confocal microscope (LSM710; Carl Zeiss Inc.Oberkochen, Germany). For live cell calcium imaging, cells were excitedwith 488 nm light from an argon laser. The emission was detected at 530nm. The cells were scanned once per second for 240 s. After 60 s ofbaseline recording in HBSS, cells were exposed to glutamate (50 μMglumatatme+10 μM glycine) in HBSS for 180 s. The intensity of thefluorescence in each cell was recorded, which represented theconcentration of intracellular calcium. The intensity of baseline wasnormalized to 1, and data were presented as folds increased than thebaseline.

Power analysis was performed by using online tool developed byStatistical Solutions LLC. According to human PET studies, a normal CBFwas averaged 22 mL and the critical neuronal damage CBF was 12-14 mLwith a variance of approximate 12 mL. We stratified each group intothree subgroups according to relative CBF (rCBF)<0.4, 0.4-0.6, and >0.6.Based on our previous studies, we needed a minimum of four mice in eachof the six subgroups to have a power of 0.8 for statistics. We also knewthat the mortality rate was about 20% in this model. Therefore, wedecided to have a total of 30 mice for the study.

Results are presented as the means±standard deviation. Regressionanalysis was performed by GraphPad-prism 5 software (GraphPad SoftwareInc. La Jolla, Calif., USA). For exponential regression, the formula ispresented as: Y=(Y0−Plateau)×exp (−K×X)+Plateau. Unpaired Student'st-test was used for comparison between two groups, while one wayanalysis of variance (ANovA) for multiple groups to determine thesignificance of the difference.

Example 34 Correlation of CBF and Infarct Volume

Perfusion MRI allows early assessment of CBF during ischemia. We usedperfusion MRI to limit variations in infarct size that were caused byvariations in cerebrovasculature. The region of interest for perfusionwas at the center of MCA territory (FIGS. 23 (a) and (b)). It wasrepresented by three 0.5 mm-thick slices. The infarct volume wasmeasured in these three slices and in all slices, while perfusion wasmeasured in the ipsilateral and contralateral hemispheres in threeslices. The rCBF was defined as ipsilateral/contralateral incorresponding regions. Infarction is caused by a reduction in CBF (FIG.23 (b)). The infarct size was correlated with the degree of reduced CBF.When the rCBF was decreased under 0.4, the infarct volume was thelargest: 29.0±5.85 mm³ in the three center slices and 105.7±21.12 mm³ inall slices. When the rCBF was between 0.4 and 0.6, the infarct volumewas 5.1±1.08 mm³ in the three center slices and 12.3±3.8 mm³ in allslices. When the rCBF was more than 0.6, no infarction was observed.

Linear regression was performed to assess infarct volume in the threecenter slices and in all slices (FIG. 23 (d 1)). The correlationcoefficient R² was high (0.9802, p<0.0001), suggesting the three centerslices could be used to represent the whole brain involved in MCAstroke. Regression analysis was performed to assess the correlationbetween CBF and infarct volume. Absolute CBF in the MCA territory wascorrelated with infarct volume in the three center slices (R²=0.9038)(FIG. 23 (c 1)). rCBF showed a slightly better correlation coefficient(R²=0.9626) (FIG. 23 (c2)). Consistently, rCBF was correlated well withinfarct volume in all slices (R²=0.9559) (FIG. 23 (c2)).

Example 35 PTx Treatment Attenuated Infarct Volume after pMCAO

The mortality rate was 16.7% in the PTx-treatment group and 13.3% in thecontrol group. During the study, two animals died from anesthesiaoverdose and one from massive stroke in the PTx-treatment group; whileone died from anesthesia overdose and one from massive stroke in thecontrol group. We excluded them from the study. The 25 survived mice (12in PTx group and 13 in the control group) were scanned with MRI tomeasure the CBF and infarction volume. Regression analysis showed thatPTx treatment shifted the exponential model predicting infarct volumebased on rCBF (FIG. 24 (a)). When the rCBF was decreased under 0.4, theinfarct volume was the largest: 105.7±21.12 mm³ in the control group(n=5) and 101.0±16.01 mm³ in PTx group (n=3, p=0.751). When the rCBF wasbetween 0.4 and 0.6, the infarct volume was reduced by PTx treatment(12.3±3.8 mm³ in the control group, n=4 and 2.12±1.58 mm³ in PTx group,n=5, p<0.01). When the rCBF was more than 0.6 (n=4 each), no lesion wasobserved in either group (FIG. 24 (b)). Absolute CBF was measured inboth ipsilateral (ischemic) and contralateral (non-ischemic) hemispheresin two groups. According to the rCBF, they were divided into threegroups: high CBF, middle CBF, and low CBF (FIG. 24 (c)). In the low CBFgroup, the absolute CBF (aCBF) in the ipsilateral hemisphere was42.9±7.8 mL/100 g/min in the control group and 37.4±4.4 mL/100 g/min inthe PTx treatment group; in the contralateral hemisphere, it was141.4±12.7 versus 134.7±25.8 mL/100 g/min. In the middle CBF group, itwas 77.2±10.6 versus 77.0±14.5 mL/100 g/min in the ipsilateralhemisphere and 151.3±19.3 versus 153.6±28.1 mL/100 g/min in thecontralateral hemisphere. In the high CBF group, it was 115.9±8.4 versus117.4±3.8 mL/100 g/min in the ipsilateral hemisphere and 174.4±6.2versus 173.3±6.4 mL/100 g/min in the contralateral hemisphere. Thus, PTxtreatment did not change the aCBF in either ischemic or non-ischemicsides, suggesting the outcome of PTx treatment as we described next wasnot a direct effect from enhanced blood flow, but a consequence ofneuroprotection in the presence of reduced blood flow.

Example 36 PTx Treatment Increased Neurons Survival after GlutamateInduced Cell Death In Vitro

Primary culture neurons were used to test whether PTx treatment couldprotect neurons against glutamate excitotoxicity as it often occursduring ischemic stroke. MTT assay showed that glutamate treatmentreduced the survival of neurons, while PTx treatment saved them. Thesurvived neurons were reduced to 0.618±0.06 after glutamate treatment(the baseline control was normalized to 1, p<0.01). When PTx was addedbefore the glutamate treatment, the survived neurons increased to1.1±0.12 (p<0.01, FIGS. 25 (a) and (b)). LDH is a soluble cytosolicenzyme that is released into the culture medium following loss ofmembrane integrity. Its release correlates with cell lysis. Glutamateincreased LDH release to 1.31±0.11 (the baseline control was normalizedto 1, p<0.01). PTx treatment reversed this increase (0.76±0.08, p<0.01,FIG. 25 (c)).

Example 37 PTx Treatment Decreased Glutamate-Induced Calcium Influx intoNeurons

Calcium imaging was performed to measure the intracellular calcium. Theintensity of fluorescence which represented the concentration ofintracellular calcium increased sharply and reached the peak quicklyafter glutamate was administered. PTx treatment slowed down this rapidincrease (T½: 18.5±2.5 s in glutamate vs. 85.4±10.2 s in PTx) (FIGS. 26(a) and (c)). The peak value represented the maximum amount of calciuminflux. It was increased 5.1±0.5 folds compared to the baseline afterglutamate stimuli, whereas PTx treatment reduced this increase (2.8±0.3folds, p<0.01, FIGS. 26 (a) and (b)).

Example 38 PTx Treatment Prevented Neurons from Apoptosis after pMCAO

Cleaved caspase-3 is a marker of apoptosis. PTx treated mice had lesscleaved caspase-3 positive cells at 24 h after pMCAO (297.5±83.6/mm² vs.677.7±117.8/mm² in control mice, p<0.01, FIGS. 27 (a) and (b)). PTx alsoreduced the density of caspase-3 positive cells (1.36±0.05 vs. 1.89±0.2in controls, p<0.01, FIGS. 26 (c) and (d)).

Example 39 Materials and Methods for Examples 40-45

All methodologies contained herein are from Z. Tang et al., CX3CR1deficiency suppresses activation and neurotoxicity ofmicroglia/macrophage in experimental ischemic stroke, J. ofNeuroinflammation 2014, 11:26. This reference is incorporated byreference in its entirety for all purposes.

Breeding pairs of CX3CR1^(−/−) mice were obtained from The JacksonLaboratory (Bar Harbor, Me., US). Knock-outs were generated by replacingthe second exon of the CX3CR1 gene with the enhanced green fluorescentprotein (GIP) reporter gene, and backcrossed for more than 10generations to C57BL/6. Cells under control of the endogenous CX3CR1locus (that is, microglia, macrophages, dendritic cells, and so forth)in homozygote CX3CR1^(−/−) mice are labeled with GFP and also lackCX3CR1 receptor function. Wild-type (WI) C57BL/6 mice were purchasedfrom Taconic (Oxnard, Calif., USA). All animals were housed inpathogen-free conditions at the animal facilities of the BarrowNeurological Institute, Phoenix, Ariz., LISA. All experimentalprocedures were approved by the Institutional Animal Care and UseCommittee of the Barrow Neurological Institute and performed accordingto the Revised Guide for the Care and Use of Laboratory Animals.

Adult male mice (aged 10 to 14 weeks, weight 24 to 27 g) were exposed totransient (90 minutes) focal cerebral ischemia induced by occlusion ofthe right middle cerebral artery using an intraluminal filament method.The production of an infarct was confirmed by 2,3,5-triphenyltetrazoliumchloride (TIC).

Daily neurological deficit assessment was performed by investigatorsblinded to the control and MCAO groups as described previously. Ratingscale: 0=no deficit, 1=failure to extend left forepaw, 2=decreased gripstrength of left forepaw, 3=circling to left by pulling the tail, and4===spontaneous circling.

Magnetic resonance imaging (MRI) was performed on a 7-T small animalMRI, 30-cm horizontal-bore magnet, and BioSpec Avance III spectrometer(Balker Daltonics Inc., Fremont, Calif., USA). In order to assesswhether the ischemia and reperfusion were induced successfully, singleslice cerebral blood flow (CBF) images were acquired before MCAO, 1 hourafter MCAO and 24 hours after reperfusion, using a Continuous ArterialSpin Labeling sequence. Multiple Segments Echo Planer Imaging sequenceswere used to acquire apparent diffusion coefficient (ADC) values 30minutes after MCAO to assess the damage volume. 12-weighted images wereacquired 24 and 72 hours after MCAO to evaluate the development ofischemic lesions, using a Rapid Acquisition with Refocused Echoessequence. MRI data were analyzed using the MED×3.4.3 software package(Medical Numerics Inc., Germantown, Mass., USA) on a LINUX workstation.

Reactive oxygen species (ROS) generated in the brain were assessed inlive mice by using the Xenogen IVIS200 imager (Caliper Life Sciences,Alameda, Calif., USA). Briefly, mice were intraperitoneally injectedwith 200 mg/kg Luminol (Invitrogen, Carlsbad, Calif., USA). After 10minutes, bioluminescence images were captured with exposure time of 3minutes. A region of interest tool was used to measure thechemiluminescent intensity of the whole brain. Data were collected asphotons per second per centimeter squared using the Living Imagesoftware (Caliper Life Sciences).

Terminally anesthetized mice were perfused intracardially with salinefollowed by 4% paraformaldehyde. The fixed brains were embedded inparaffin and cut into serial 6 μm thick coronal slides.Immunohistochemistry was performed with antibodies against Iba-1 (WakoChemicals USA Inc., Richmond, Va., USA) to identify macrophages andmicroglia; 4-hydroxy-2-nonenal (4-HNE; Abcam, Cambridge, Mass., USA) and8-hydroxy-2-deoxyguanosine (8-OHdG; Abeam) to identify lipidperoxidation and damaged DNA of oxidative impairment. Immunolabeling wasdetected by applying the peroxidase-antiperoxidase procedure with3,3′-diaminobenzidine as a co-substrate. For double immunofluorescentstaining, antibodies against NeuN Billerica, Mass., USA) and cleavedCaspase-3 (Cell Signaling, Danvers, Mass., USA) were used to identifyapoptotic neurons. Respective negative controls that omit primaryantibodies and positive controls were applied for each case. Thepositive cells were counted at 20× magnification in matched sections.Results are presented as Iba-1⁺, 4-HNE⁺, 8-OHdG⁺ orcleaved-Caspase-3⁺/NeuN⁺ cells per mm² within areas measured from 20images using Image J. 1.34vi software (National Institutes of Health).

Brain homogenates were prepared from WT and CX3CR1^(−/−) mice 72 hoursafter MCAO. Animals were anesthetized and the brains were removed andimmediately frozen in liquid nitrogen. The ipsilateral or contralateralhemisphere was homogenized in RIPA buffer (10 μl/mg brain, Sigma, St.Louis, Mo., USA). Protein concentration was measured with bicinchoninicacid Protein Assay kit (Pierce, Appleton, Wis., USA). The total proteinconcentration was adjusted to 1 mg/ml protein extract. Theconcentrations of IL-10, IL-6, and TNF-α in brain homogenates werequantified by enzyme-linked immunosorbent assay (ELISA) kits (BioLegend,San Diego, Calif., USA) and converted into pg/mg protein extract.

In order to visualize proliferating mononuclear cells in CNS, mice wereinjected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU) (50 μg/gof mouse weight in saline, Sigma) immediately before the MCAO procedureand again 24, 48, and 60 hours after surgery. At 72 hours after surgery,mice were anesthetized with isoflurane and transcardially perfused withPBS. Brains were removed, and microglia and invading leukocyte isolationwas performed according to the standardized protocol described below.

The isolation of microglia and invading leukocytes are based ondiscontinuous percoll gradients. Briefly, fresh brain tissues wereremoved from mice and cut into ˜2 mm pieces and incubated in 10 mMHepes/NaOH buffer (10 mM HEPES, 150 mM NaOH, 7 mM KCL, 1 mM MgCl₂, 1 mMMgCl₂, 0.36 mM CaCl₂)) containing 1 mg/ml collagenase (Sigma) for 1 hourat 37° C. The tissues were dispersed with a syringe, filtered through a100-mm wire mesh, and centrifuged at 2,000 rpm for 5 minutes at 4° C.After centrifugation, cell pellets were resuspended in 15 ml 30% Percoll(Amersham Biosciences, Piscataway, N.J., USA), and centrifuged against70% Percoll in a 50-ml tube for 15 minutes. The cell monolayer at the 30to 70% Percoll interface was collected and washed once for furtherstaining.

The number of microglia/macrophage, their proliferation properties andinflammatory cytokine secretion were analyzed by flow cytometry. Singlecell suspensions prepared from brain tissues were stained withfluorescently labeled antibodies: APC-CD45, PerCP-Cy5.5-CD45, PE-Ly6G,BV421-Ly6G, PE-Cy7-CD11b, PerCP-Cy5.5-BrdU, PE-IL-1β, V450-IL-6, orAPC-TNF-α at designed combination. All antibodies and the isotypecontrols were purchased from BD Biosciences, San Jose, Calif., USA. Thestaining was performed according to the manufacturer's instructions.After staining, samples were analyzed using a FACSAria I flow cytometer(BD Biosciences). To avoid the interference of GFP expressed onCX3CR1^(−/−) microglia/macrophage, fluors such as FITC and Alexafluor488 that excite in the same spectrum at the same filter sets as GFP wereexcluded and the proper compensation was performed in flow cytometry.Subsequent data analyses were completed using FACSDiva software or FCSExpress 4 software (BD Biosciences).

Microglia/macrophages were enriched from the single-cell suspension ofthe ischemic hemisphere of MCAO mice using CD11b MicroBeads (MiltenyiBiotec, Auburn, Calif., USA), and then sorted viaAPC-45/PE-Cy7-CD11b/BV421-Ly6G makers from the remaining cells byFACSAria I using Diva software (BD Biosciences). Purity of microglia andmacrophages obtained by this approach reached 99%.

Total RNA was extracted from the sorted microglia/macrophages using theRNeasy Micro Kit (Qiagen, Germantown, Md., USA) according to themanufacturer's instructions; 1 μg was used to synthesize the firststrand of cDNA using the Superscript First-Strand Synthesis System forreal-time PCR (Invitrogen). PCR was performed on the Opticon 2 Real-TimePCR Detection System (Bio-Rad, Hercules, Calif., USA) usingcorresponding primers (Table 1) and iQ™ SYBR Green Supermix (Bio-Rad).Cycle conditions included heating for 5 minutes at 95° C., followed by40 cycles of 30 seconds at 95° C., 30 seconds at 60° C., and 60 secondsat 72° C. A melt curve analysis was performed to ensure specificamplification. For each target gene, relative levels of expression werenormalized against housekeeping gene GAPDH of the same sample. Therelative expression levels of the mRNAs were then reported as foldchanges versus sham controls.

Results are presented as the means±SEM. Statistical differences betweentwo groups were evaluated by the two-tailed unpaired Student's t-test.Multiple comparisons were performed with two-way analysis of varianceaccompanied by Bonferroni post-hoc test. Values of P<0.05 wereconsidered significant.

Example 40 Reduction of Infarct Volume and Neurological Deficit byCx3CR1 Deficiency after Middle Cerebral Artery Occlusion

To ensure the success of the MCAO model, CBF of right middle cerebralartery territory was examined at 24 hours before MCAO, and 1 hour and 24hours following MCAO using high-field MRI. Animals wherein ischemia andreperfusion were induced successfully were selected for furtheranalysis. No significant difference in CBF at baseline (WT, 171.3±9.5 vsCX3CR1^(−/−), 174.2±10.1 ml/100 g/min, P>0.05), ischemia (WT, 33.5±14.3vs CX3CR1^(−/−), 31.2±13.1 ml/100 g/min, P>0.05) and reperfusion (WT,160.1±15.1 vs CX3CR1^(−/−), 155±12.3 ml/100 g/min, P>0.05) was observedbetween WT and CX3CR1^(−/−) mice. Furthermore, ADC (measuring themagnitude of diffusion of water molecules within cerebral tissue) wasacquired 30 minutes after MCAO to assess the damage volume ofipsilateral hemisphere. No significant differences in damage volume wereobserved between CX3CR1^(−/−) and WT mice (WT, 122.2±8.3 mm³;CX3CR1^(−/−), 119.1±6.7 mm³).

To assess the infarct volume, T2-weighted images were acquired at 24 and72 hours following MCAO. The infarct volume in WT mice was 36.5±5.7 mm³at 24 hours and increased to 45.8±6.8 mm³ at 72 hours. WithinCX3CR1^(−/−) mice, infarct volume was 26.9±5.7 mm³ at 24 hours, and amodest but significant decrease (to 19.0±4.9 mm³) was observed at 72hours (FIG. 28 (A)). Although there was no significant difference ininfarct volume at 24 hours between the two groups, CX3CR1^(−/−) miceshowed markedly smaller infarct volume at 72 hours relative to WT mice(P<0.01, FIG. 28 (B)). The infarct observed in MRI scan was confirmed byTTC staining.

To further assess the differential response to MCAO in CX3CR1^(−/−) andWT mice, the neurological deficit was assessed daily following MCAO. WTmice had an average clinical score of 2.4±0.4 at 24 hours and 2.0±0.3 at72 hours, while CX3CR1^(−/−) mice had clinical scores of 2.0±0.3 at 24hours and 1.2±0.2 at 72 hours indicative of the beginning of recovery at72 hours in CX3CR1^(−/−) mice but not in WT mice (FIG. 28 (C)).

Example 41 CX3CR1 Deficiency Attenuates Neuronal Apoptosis after MiddleCerebral Artery Occlusion

Double staining with NeuN (neuron marker) and cleaved Caspase-3(apoptotic marker) was performed to investigate whether CX3CR1-dependentdifferences observed in the size of ischemic damage at 72 hoursfollowing MCAO were associated with differential neuronal apoptosis inperi-infarct areas. More cleaved. Caspase-3 positive cells were observedin WT mice compared to CX3CR1^(−/−) mice and co-localized with NeuN inmost apoptotic cells (FIG. 29 (A)). The number of cleaved Caspase-3positive neurons was 179.3±15. /mm′ in WT mice and 95.1±16.9/mm² inCX3CR1±mice 72 hours after MCAO (P<0.01, FIG. 29 (B)). To reveal thephenotype of the other apoptotic cells, double staining of cleavedCaspase-3 with Iba-1 or GFAP markers, respectively, were employed. Theseexperiments revealed cleaved Caspase-3 positive staining within somemicroglia and astrocytes (data not shown).

Example 42 Fewer Microglia and Macrophages in Ipsilateral Hemisphere ofCX3CR1−/− after Middle Cerebral Artery Occlusion

Microglia activation plays an important role in the pathologicalprogression after stroke, and is regulated by CX3CR1. To investigate theeffects of CX3CR1 deficiency upon these processes, expression ofmicroglia/macrophage activation marker Iba-1 was examined at differentsites of the brain in WT and CX3CR1^(−/−) mice 72 hours following MCAO(FIG. 30 (A)). In the ipsilateral hemisphere of WT mice, the numbers ofIba-1 positive cells in hippocampus, striatum, cortex and peri-infractarea (666.1±50.0, 1132.8±96.1, 730.9±68.1, 489.4±56.1/mm²) weresignificantly higher than those in CX3CR1^(−/−) mice (424.6±43.1,492.7±42.3, 230.1±20.1, 262.5±29.8/mm²) (FIG. 30 (B)). In thecontralateral hemisphere, the numbers of Iba-1 positive cells in thehippocampus, striatum and cortex were similar (307.9±15.5 vs 316.0±14.6,110.2±11.5 vs 111.8±9.7, 94.0±8.6 vs 92.4±13.1, WT vs CX3CR1^(−/−))regardless of genotype (FIG. 30 (B)).

To further distinguish microglia from macrophages within the Iba-1positive cell population, flow cytometry was used to delineateCD45^(low)/CD11b⁺/Ly6G⁻ (microglia) and CD45^(hi)/CD11b⁺/Ly6G⁻(macrophage/activated microglia) sub-populations within ischemic lesionsof WT and CX3CR1^(−/−) mice. As shown in FIGS. 30, (C) and (D), moremacrophages/activated microglia infiltrated in the ischemia lesions inWT mice compared to CX3CR1^(−/−) mice (34.1±2.5% vs 15.9±1.8%). Inaddition, WT mice displayed more CD45^(low) microglia than CX3CR1^(−/−)mice (22.1±2.3% vs 11.7±2.1%, FIGS. 30 (C) and (D)). Both cellpopulations were similar in WT and CX3CR1^(−/−) mice in the control(non-ischemic) contralateral hemisphere (FIGS. 30 (C) and (D)).

Following ischemia, activated microglia/macrophage can potentially exerteither a protective or detrimental effect, suggesting that these cellsmay acquire different phenotypes belonging to the classical (M1) or tothe alternative (M2) active status. We found that activated M1-likeIba-1⁺ cells, which have shorter and thicker processes and bigger cellbodies, were visualized in the WT brain section, while ramified M2-likeIba-1⁺ cells were predominantly located in the CX3CR1^(−/−) brain (FIG.30 (A)). To evaluate their M1/M2 polarization, we sorted and purifiedmicroglia/macrophages (CD45⁺/CD11b⁺/Ly6G⁻) from the ischemic hemisphereof CX3CR1^(−/−) and WT MCAO mice brain. Using real-time PCR, we foundthat the levels of tested M1- and M2-type genes in FluorescenceActivated Cell Sorter (FACS)-sorted microglia/macrophages from WT micewere increased starting from 1 day after MCAO and further elevated by 3days post-MCAO (data not shown). Within CX3CR1^(−/−)microglia/macrophages, the M2-type genes (Yml, Mcrl) were significantlyincreased, whereas the M1-type gene (iNOS) was notably decreasedcompared to WT microglia/macrophages when isolated at 72 hours afterMCAO (FIG. 30 (E)). These results suggest that deficiency of CX3CR1 mayfacilitate the alternative activation (M2 state) ofmicroglia/macrophages in stroke.

Example 43 Reduced Proliferation of Macrophages and Microglia inIpsilateral Hemisphere of CX3CR1−/− Mice after Middle Cerebral ArteryOcclusion

To determine whether the decrease in CD11b⁺Ly6G⁻ cells observed inischemic lesions of CX3CR1^(−/−) MCAO mice, in addition to decreasedchemotaxis of monocytes, was due to suppressed expansion ofmicroglia/macrophage, we assessed proliferation of CD11b⁺Ly6G⁻ cells.Proliferation analysis was performed on populations ofCD45^(low)/CD11b⁺/Ly6G⁻ cells (microglia), and CD45^(hi)/CD11b⁺/Ly6G⁻cells (macrophage/activated microglia) obtained from CX3CR1^(−/−) and WTmice injected with BrdU (FIG. 31 (A)). Quantitative analysis revealed asignificant 3-fold increase in the number ofCD45^(low)/CD11b⁺/Ly6G⁻/BrdU⁺ cells in the ipsilateral relative to thecontralateral hemisphere in WT mice 72 hours after stroke (FIG. 31 (B)).A significant reduction (71.1%) in the number of proliferatingCD45^(low)/CD11b⁺/Ly6G⁻ cells was observed in the ipsilateral hemispherein CX3CR1^(−/−) compared to WT mice (WT, 43.3±4.1%; CX3CR1^(−/−),12.5±2.2%, P<0.01, FIG. 31 (B)). There were no significant differencesin the numbers of CD45^(low)/CD11b⁺/Ly6G⁻/BrdU⁺ cells in thecontralateral hemisphere between the two experimental groups, althoughCX3CR1^(−/−) mice showed a lesser trend (WT, 11.5±2.9%; CX3CR1^(−/−),6.7±0.7%, P>0.05, FIG. 31 (B)). We also observed a modest butsignificant reduction (23.2%) in the number of proliferatingCD45^(hi)/CD11b⁺/Ly6G⁻ cells in the ipsilateral hemisphere inCX3CR1^(−/−) compared with WT mice (WT, 42.1±2.7%; CX3CR1^(−/−),32.3±3.9%, P<0.05). Collectively, these results indicate that CX3CR1deficiency affects ischemic injury-induced proliferation of residentmicroglia and recruited macrophages.

Example 44 CX3CR1 Deficiency Attenuates Reactive Oxygen SpeciesGeneration in Brain after Middle Cerebral Artery Occlusion

ROS, generated as by-products of cellular metabolism, have long beenknown to be a component of the inflammatory response after ischemia. Toinvestigate whether CX3CR1 deficiency had effects upon ROS production,we assessed ROS levels in live mice using the Xenogen IVIS200 imager at24 and 72 hours after MCAO. The chemiluminescence detection of ROS wasperformed in the brains, specifically in the ipsilateral hemisphere(FIG. 32 (A)). The mean chemiluminescence intensity of brain in WT micewas 1,090.3±127.4 p/s/cm²/sr (photons per second per centimeter squaredper steradian) at 24 hours and 850.9±81.7 p/s/cm²/sr at 72 hours. WithinCX3CR1^(−/−) mice, mean chemiluminescence intensity values were1,350.9±118.9 p/s/cm²/sr at 24 hours and 641.7±47.6 p/s/cm²/sr at 72hours. At 24 hours post-ischemia, no differences in ROS levels wereobserved between the two groups (P>0.05). At 72 hours post-ischemia, theROS levels decreased significantly in CX3CR1^(−/−) mice compared to 24hours (P<0.01), while no change was observed in WT mice (P>0.05) (FIG.32 (B)). In addition, the oxidative impairment of neurons wasimmunohistochemically assessed by stain for lipid peroxidation with4-HNE and damaged DNA with 8-OHdG (FIG. 32 (C)). The number of stainedcells in the CX3CR1^(−/−) mice was notably less compared with WT mice(P<0.01) (FIG. 32 (D)).

Example 45 CX3CR1 Deficiency Impairs Inflammatory Signaling in Microgliaand Macrophage in Ischemic Brain

To determine whether CX3CR1 deficiency is associated with changes in theexpression of inflammatory mediators produced by activated macrophagesand microglia, ELISA was used to screen injured brain homogenates fordifferences in cytokine production. Consistent with previous reports,expression of a subset of inflammatory cytokines IL-6, and TNF-α) wasincreased after MCAO regardless of genotype. In WT mice, the amounts ofIL-10, IL-6, and TNF-α were 160.9±8.4, 56.5±6.1, 253.0±22.9 pg/mg in theipsilateral hemisphere and 80.7±4.1, 19.2±2.2, 101.9±15.3 pg/mg in thecontralateral hemisphere, respectively. In CX3CR1^(−/−) mice, theamounts of IL-10, IL-6, and TNF-α were 94.2±11.9, 29.0±5.1, 191.9±19.8pg/mg in the ipsilateral hemisphere and 74.5±5.1, 15.5±1.1, 116.5±13.7pg/mg in the contralateral hemisphere, respectively. Notably,post-injury expressions of these cytokines were markedly reduced inCX3CR1^(−/−) mice in the ipsilateral hemisphere, while no difference wasobserved in the contralateral hemisphere (FIG. 33 (A)).

To determine whether deficient CX3CR1 signaling in CNSmicroglia/macrophages could account for these cytokine expressionchanges, a series of controlled ex vivo flow cytometry assays wereperformed. Microglia and macrophages were isolated from ischemic brainsof WT and CX3CR1^(−/−) mice, followed by quantification ofIL-1β⁺/CD11b⁺/Ly6G⁻, IL-6⁺/CD11b⁺/Ly6G⁻, and TNF-α⁺/CD11b⁺/Ly6G⁻ cellsusing low cytometry. Using this approach, an increased expression ofIL-1β, IL-6, and TNF-α CD11b⁺Ly6G⁻ cells (fluorescent intensity in FIG.33 (B)) as well as the numbers of cytokine-expressing CD11b⁺Ly6G⁻ cells(event quantification in FIG. 33 (C)) were detected 72 hours after MCAOin the ischemic lesions in WT mice (ipsilateral vs contralateral). Thisresponse was noticeably absent from the injured brain of CX3CR1^(−/−)mice (FIGS. 33, (B) and (C)). In the ipsilateral hemisphere,CX3CR1^(−/−) mice displayed significant reduction in expression ofIL-1β, IL-6, and TNF-α in CD11b⁺Ly6G⁻ cells and cytokine-expressingCD11b⁺Ly6G⁻ cell numbers (P<0.05 vs WT, FIGS. 33, (B) and (C)).

The various methods and techniques described above provide a number ofways to carry out the invention. Of course, it is to be understood thatnot necessarily all objectives or advantages described may be achievedin accordance with any particular embodiment described herein. Thus, forexample, those skilled in the art will recognize that the methods can beperformed in a manner that achieves or optimizes one advantage or groupof advantages as taught herein without necessarily achieving otherobjectives or advantages as may be taught or suggested herein. A varietyof advantageous and disadvantageous alternatives are mentioned herein.It is to be understood that some preferred embodiments specificallyinclude one, another, or several advantageous features, while othersspecifically exclude one, another, or several disadvantageous features,while still others specifically mitigate a present disadvantageousfeature by inclusion of one, another, or several advantageous features.

Furthermore, the skilled artisan will recognize the applicability ofvarious features from different embodiments. Similarly, the variouselements, features and steps discussed above, as well as other knownequivalents for each such element, feature or step, can be mixed andmatched by one of ordinary skill in this art to perform methods inaccordance with principles described herein. Among the various elements,features, and steps some will be specifically included and othersspecifically excluded in diverse embodiments.

Although the invention has been disclosed in the context of certainembodiments and examples, it will be understood by those skilled in theart that the embodiments of the invention extend beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses andmodifications and equivalents thereof.

Many variations and alternative elements have been disclosed inembodiments of the present invention. Still further variations andalternate elements will be apparent to one of skill in the art. Amongthese variations, without limitation, are the selection of constituentmodules for the inventive compositions, and the diseases and otherclinical conditions that may be diagnosed, prognosed or treatedtherewith. Various embodiments of the invention can specifically includeor exclude any of these variations or elements.

In some embodiments, the numbers expressing quantities of ingredients,properties such as concentration, reaction conditions, and so forth,used to describe and claim certain embodiments of the invention are tobe understood as being modified in some instances by the term “about.”Accordingly, in some embodiments, the numerical parameters set forth inthe written description and attached claims are approximations that canvary depending upon the desired properties sought to be obtained by aparticular embodiment. In some embodiments, the numerical parametersshould be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and parameters setting forth the broad scopeof some embodiments of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspracticable. The numerical values presented in some embodiments of theinvention may contain certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment ofthe invention (especially in the context of certain of the followingclaims) can be construed to cover both the singular and the plural. Therecitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided with respectto certain embodiments herein is intended merely to better illuminatethe invention and does not pose a limitation on the scope of theinvention otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember can be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. One ormore members of a group can be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is herein deemed to contain the groupas modified thus fulfilling the written description of all Markushgroups used in the appended claims.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations on those preferred embodiments will become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Itis contemplated that skilled artisans can employ such variations asappropriate, and the invention can be practiced otherwise thanspecifically described herein. Accordingly, many embodiments of thisinvention include all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and printedpublications throughout this specification. Each of the above citedreferences and printed publications are herein individually incorporatedby reference in their entirety.

In closing, it is to be understood that the embodiments of the inventiondisclosed herein are illustrative of the principles of the presentinvention. Other modifications that can be employed can be within thescope of the invention. Thus, by way of example, but not of limitation,alternative configurations of the present invention can be utilized inaccordance with the teachings herein. Accordingly, embodiments of thepresent invention are not limited to that precisely as shown anddescribed.

The invention claimed is:
 1. A method of increasing expression ofvascular endothelial growth factor (VEGF) to treat multiple sclerosis,in a subject having multiple sclerosis, the method comprising the stepsof: administering a therapeutically effective dosage of a compositioncomprising pertussis toxin (PTx) comprising subunits A and B to thesubject.
 2. The method of claim 1, wherein the composition isadministered intracerebroventricularly (icy) or intraperitoneally (ip).3. The method of claim 1, further comprising inhibiting migration of Tcells into neurologically damaged tissue via administration of thecomposition.
 4. The method of claim 1, wherein the composition isadministered to the subject in conjunction with a G-protein, chemokineand/or adhesion blocking agents.
 5. The method of claim 1, furthercomprising increasing blood vessel density in the subject's brain cortexand/or spinal gray matter via administration of the composition.
 6. Themethod of claim 1, wherein the therapeutically effective dosagecomprises at least 500 ng PTx.
 7. The method of claim 1, wherein thetherapeutically effective dosage comprises at least 1000 ng PTx.
 8. Themethod of claim 1, wherein the therapeutically effective dosagecomprises at least 2000 ng PTx.
 9. The method of claim 1, wherein thetherapeutically effective dosage comprises at least 3000 ng PTx.
 10. Themethod of claim 1, wherein the therapeutically effective dosage isbetween 500 ng/day and 1000 ng/day PTx.
 11. The method of claim 1,wherein the therapeutically effective dosage is between 1000 ng/day and2000 ng/day PTx.